USO08080578B2
(12) United States Patent (10) Patent No.: US 8,080,578 B2 Liggett et al. (45) Date of Patent: *Dec. 20, 2011
(54) METHODS FOR TREATMENT WITH 5,998.458. A 12/1999 Bristow ...... 514,392 BUCNDOLOL BASED ON GENETIC 6,004,744. A 12/1999 Goelet et al...... 435/5 6,013,431 A 1/2000 Söderlund et al. 435/5 TARGETING 6,156,503 A 12/2000 Drazen et al...... 435/6 6,221,851 B1 4/2001 Feldman ... 51446 (75) Inventors: Stephen B. Liggett, Clarksville, MD 6,316,188 B1 1 1/2001 Yan et al...... 435/6 6,365,618 B1 4/2002 Swartz ... 514,411 (US); Michael Bristow, Englewood, CO 6,498,009 B1 12/2002 Liggett ...... 435/6 (US) 6,566,101 B1 5/2003 Shuber et al. . 435,912 6,586,183 B2 7/2003 Drysdale et al...... 435/6 (73) Assignee: The Regents of the University of 6,784, 177 B2 8/2004 Cohn et al. . 514,248 Colorado, a body corporate, Denver, 6,797.472 B1 9/2004 Liggett ...... 435/6 6,821,724 B1 1 1/2004 Mittman et al. ... 435/6 CO (US) 6,861.217 B1 3/2005 Liggett ...... 435/6 7,041,810 B2 5/2006 Small et al...... 435/6 (*) Notice: Subject to any disclaimer, the term of this 7, 195,873 B2 3/2007 Fligheddu et al. ... 435/6 patent is extended or adjusted under 35 7,211,386 B2 5/2007 Small et al...... 435/6 7,229,756 B1 6/2007 Small et al...... 435/6 U.S.C. 154(b) by 280 days. 7,449,292 B2 11/2008 Liggett et al. . ... 435/6 This patent is Subject to a terminal dis 7,572,603 B2 8/2009 Small et al. ... 435,912 7,642,052 B2 1/2010 Small et al...... 435/6 claimer. 7,678,824 B2 * 3/2010 Liggett et al. . 514,415 2001/0016338 A1 8/2001 Snapir et al. . 435/69.1 (21) Appl. No.: 11/838,131 2001/0051712 A1 12/2001 Drysdale et al. 536,235 2002/0187491 A1 12/2002 Johnson ...... 435/6 (22) Filed: Aug. 13, 2007 2003/0039979 A1 2/2003 Liggett et al. . ... 435/6 2003/009 1998 A1 5/2003 Drysdale et al...... 435/6 Prior Publication Data 2003/01 13725 A1 6/2003 Small et al...... 536,231 (65) 2003/0124636 A1 7/2003 Leyland-Jones .. 435/7.92 US 2008/OO96982 A1 Apr. 24, 2008 2003/0143608 A1 7/2003 Filigheddu et al...... 435/6 2003/02331 18 A1 12/2003 Hui ...... 606,201 2004/0023967 A1 2/2004 Cohn et al. . 514,248 Related U.S. Application Data 2004/0033524 A1 2/2004 Johnson ...... 435/6 (62) Division of application No. 1 1/226,908, filed on Sep. (Continued) 14, 2005. (60) Provisional application No. 60/609.689, filed on Sep. FOREIGN PATENT DOCUMENTS 14, 2004, provisional application No. 60/610,706, EP O329822 6, 1994 filed on Sep. 17, 2004. (Continued) (51) Int. C. OTHER PUBLICATIONS A6 IK3I/405 (2006.01) A6 IK 47/00 (2006.01) Borjesson et al., “A novel polymorphism in the gene coding for the A6IP 43/00 (2006.01) beta 1-adrenergic receptor associated with Survival in patients with CI2O I/68 (2006.01) heart failure, Eur Heart J., 21:1853-1858, 2000. C7H 2L/02 (2006.01) Bouzamondo et al., “Beta-blocker treatment in heart failure.' Fun C7H 2L/04 (2006.01) damental & Clinical Pharmacology, 15:95-109, 2001. GOIN33/53 (2006.01) Bristow et al. “Selective versus nonselective beta-blockade for heart failure therapy: are there lessons to be learned from the COMET (52) U.S. C...... 514/415: 514/789; 435/6.1; 435/6.11; trial?” J Card Fail. 9:444-53, 2003. 435/6.17; 435/7.1:536/23.5; 536/24.31; 530/350 Bristow et al., “Effect of baseline or changes in adrenergic activity on (58) Field of Classification Search ...... None clinical outcomes in the beta-blocker evaluation of Survival trial.” See application file for complete search history. Circulation. 110:1437-42, 2004. Bristow et al., “The role of third-generation beta-blocking agents in (56) References Cited chronic heart failure.” Clinical Cardiology, 21 (Suppl I):I-3-I-13. 1998. U.S. PATENT DOCUMENTS (Continued) 4,234,595 A 11/1980 Kreighbaum et al...... 424/274 4,873,191 A 10/1989 Wagner et al...... 435/1723 Primary Examiner — Carla Myers 4,965,188 A 10, 1990 Mullis et al. . ... 435/6 5,002,867 A 3, 1991 Macevicz ...... 435/6 (74) Attorney, Agent, or Firm — Fulbright & Jaworski L.L.P. 5,130,238 A 7, 1992 Malek et al. . 435/91 5,169,766 A 12/1992 Schuster et al. 435/91 (57) ABSTRACT 5,202,231 A 4, 1993 Drmanac et al. ... 435/6 5,302,509 A 4, 1994 Cheeseman ...... 435/6 The present invention concerns the use of methods for evalu 5,595,880 A 1/1997 Weinshank et al. . 435/721 ating bucindolol treatment for a patient, particularly one with 5,648,482 A 7/1997 Meyer ...... 536, 24.33 heart failure. It concerns methods for determining whether to 5,679,524. A 10, 1997 Nikiforov et al...... 435/6 5,766,851 A 6, 1998 Shuldiner et al...... 435/6 administer or prescribe bucindolol to a patient based on 5,846,710 A 12/1998 Bajaj...... 435/6 whether the patient is homozygous for the Arg 389 polymor 5,856,092 A 1/1999 Dale et al. 435/6 phism in the B1-adrenergic receptor (AR). 5,888,819 A 3, 1999 Goelet et al. . 435/5 5,981,174 A 11/1999 Wolfetal...... 435/6 28 Claims, 29 Drawing Sheets US 8,080,578 B2 Page 2
U.S. PATENT DOCUMENTS Gene Card for ADRA2CO2c-AR available via url:
Packer et al., “Effect of carvedilol on the morbidity of patients with Kannell et al., “Prevalence, incidence, prognosis, and predisposing severe chronic heart failure.” Circulation, 106:2194-2199, 2002. conditions for atrial fibrillation: population-based estimates. Am. J. Packer et al., “The effect of carvedilol on morbidity and mortality in Cardiol. 82(8A):2N-9N, 1998. patients with chronic heart failure." N Engl J Med. 334:1349-55, McLeodet al., “Differentiation of hemodynamic, humoral and meta 1996. bolic responses to beta 1- and beta 2-adrenergic stimulation in man Perez et al., “Beta 1-adrenergic receptor polymorphisms confer dif using atenolol and propranolol.” Circulation, 67: 1076-1084, 1983. ferential function and predisposition to heart failure.” Nat Med. Office Communication, issued in European Patent Application No. 9(10): 1300-5, 2003. 05814066.6, mail date Oct. 24, 2007. Poole-Wilson et al., “Comparison of carvedilol and metoprolol on clinical outcomes in patients with chronic heart failure in the Office Communication, issued in Russian Patent Application No. Carvedilol or Metoprolol European Trial (COMET): randomised 2007 114060/13, mail date Mar. 27, 2009. controlled trial. Lancet, 362:7-13 2003. Office Communication, issued in U.S. Appl. No. 1 1/226.908, mail Port and Bristow. "B-adrenergic receptors, transgenic mice, and date Apr. 28, 2009. pharmacological model systems.” Molecular Pharmacology, Office Communication, issued in U.S. Appl. No. 1 1/226.908, mail 60(4):629-631, 2001. date Apr. 8, 2009. RathZet al., "Hierarchy of polymorphic variation and desensitization Office Communication, issued in U.S. Appl. No. 1 1/226.908, mail permutations relative to beta 1 and beta2-adrenergic receptor signal date Nov. 21, 2008. ing.” J Biol Chem, 278: 10784-10789, 2003. Office Communication, issued in U.S. Appl. No. 1 1/226.908, mail Robinson and Bristow, "Beta blockers.” Chapter 5 in: The date May 20, 2008. Pharmacologic Management of Chronic Heart Failure, The Univer Office Communication, issued in U.S. Appl. No. 1 1/226.908, mail sity of Colorado Cardiovascular Institute, pp. 57-81, 2005. date Feb. 15, 2008. Sallach and Goldstein. “Use of beta-blockers in congestive heart Office Communication, issued in U.S. Appl. No. 1 1/226.908, mail failure. Ann Med., 35:259-266, 2003. date Aug. 6, 2007. Schwartz and Turner, "Pharmacogenetics of antihypertensive drug Office Communication, issued in U.S. Appl. No. 1 1/838,142, mail responses.” Am J Pharmacogenomics, 4:151-60, 2004. date Apr. 3, 2009. Small et al., “A four amino acid deletion polymorphism in the third PCT International Preliminary Report on Patentability, issued in intracellular loop of the human a -adrenergic receptor confers International application No. PCT/US2005/032901, mail date Mar. impaired coupling to multiple effectors.” The Journal of Biological 29, 2007. Chemistry, 275(30):23059-23064, 2000. PCT International Search Report and Written Opinion, issued in Small et al., “Pharmacology and physiology of human adrenergic International application No. PCT/US2005/032901, mail date Sep. receptor polymorphisms,” Annu Rev Pharmacol Toxicol, 43:381 26, 2005. 411, 2003. SEC Filing: Registration Statement on Form S-4, dated Oct. 30, Small et al., “Polymorphisms of cardiac presynaptic O2 adrenergic 2008. receptors: diverse intragenic variability with haplotype-specific func SEC Filing: Amendment No. 1 to Registration Statement on Form tional effects. PNAS, 101: 13020-13025, 2004. S-4, dated Nov. 21, 2008. Small et al., “Synergistic polymorphisms of beta1- and alpha2C The Beta-Blocker Evaluation of Survival Trial Investigators, "A trial adrenergic receptors and the risk of congestive heart failure." N Engl of the beta-blocker bucindolol in patients with advanced chronic J Med, 347: 1135-42, 2002. heart failure.” N. Engl. J. Med., 344(22): 1659-1667, 2001. Sofowora et al., “A common beta 1-adrenergic receptor Workman et al., “Chronic beta-adrenoceptor blockade and human polymorphism (Arg389Gly) affects blood pressure to beta-block atrial cell electrophysiology: evidence of pharmacological remodel ade. Clin Pharmacol Ther, 73:366-71, 2003. ling.” Cardiovascular Research, 58:518-525, 2003. Terra et al., “beta-Adrenergic receptor polymorphisms and responses Office Communication, issued in Australian Patent Application No. during titration of metoprolol controlled release? extended release in 2005284822, dated Mar. 1, 2010. heart failure. Clin Pharmacol Ther, 77: 127-37, 2005. “Introduction to O-adrenoreceptors.” located online at http://www. The BEST Steering Committee, “Design of the beta-blocker evalu adrenoceptor.com/alphaintro.htm, printed on Apr. 7, 2010. ation survival trial (BEST).” Am J Cardiol. 75:1220-3, 1995. Adolfsson et al., "Characterization of O-adrenoceptor Subtypes in Torp-Pedersen et al., “The incomplete bucindolol evaluation in acute pregnant human myometrium.” Gynecol. Obstet. Invest. 45:145 myocardial infarction trial (BEAT).” The European Journal of Heart 150, 1998. Failure, 4:495-499, 2002. Arner, "Adrenergic receptor function in fat cells.” Am. J. Clin. Nutr. Wedel et al., "Challenges of Subgroup anaylses in multinational 55:228S-236S, 1992. clinical trials: experiences from MERIT-HF trial.” Am Heart J. Baldwin et al., “Identification of a polymorphic glutamic acid stretch 142:502-511, 2001. in the O-adrenergic receptor and lack of linkage with essential White et al., “An evaluation of the beta-1 adrenergic receptor hypertension.” Journal of the American Society of Hypertension, Arg389Gly polymorphism in individuals with heart failure: a 12:853-857, 1999. MERIT-HF sub-study.” Eur.J Heart Fail. 5:463-8, 2003. Barany, "Genetic disease detection and DNA amplification using Wollert and Drexler, "Carvedilol prospective randomized cumulative cloned thermostable ligase.” Proc. Natl. Acad. Sci. USA, 88:189-193, survival (COPERNICUS) trial.” Circulation, 106:2164-2166, 2002. 1991. Bettoni and Zimmermann, 'Autonomic tone variations before the Bengtsson, “Polymorphism in the B1-adrenergic receptor gene and onset of paroxysmal atrial fibrillation.” Circulation, 105:2753-2759, hypertension.” Circulation, 104(2): 187-190, 2001. 2002. Berrettini and Hoehe, “A polymorphism of the beta adrenergic Blumenfeld et al., “Beta-adrenergic receptor blockade as a therapeu receptor gene (BADR) detected with Bgl I.” Nucleic Acids Res., tic approach for Suppressing the renin-angiotensin-aldosterone sys 16:7754, 1988. tem in normotensive and hypertensive Subjects.” Am J Hypertens, Björklund et al., “O2-adrenoceptor-overexpressing mice are 12(5):451–459, 1999. impaired in executing nonspatial and spatial escape strategies. Mol. Chen et al., “Initiation of atrial fibrillation by ectopic beats originat Pharmacol., 54:569-576, 1998. ing from the pulmonary veins: electrophysiological characteristics, Blum et al., “Molecular mechanism of slow acetylation of drugs and pharmacological responses, and effects of radiofrequency ablation.” carcinogens in humans.” Proc. Natl. Acad. Sci. USA, 88.5237-5241, Circulation, 100: 1879-1886, 1999. 1991. Flaa et al., “Sympathetic activity and cardiovascular risk factors in Böhm et al., “Desensitization of adenylate cyclase and increase of young men in the low, normal, and high blood pressure ranges.” G, in cardiac hypertrophy due to acquired hypertension.” Hyperten Hypertension, 47:396–402, 2006. sion, 2001): 103-112, 1992. Julius and Majahalme, “The changing face of sympathetic overactiv Böhm et al., “Increase of G, in human hearts with dilated but not ity in hypertension.” Ann. Med., 32:365-370, 2000. ischemic cardiomyopathy.” Circulation, 82:1249-1265, 1990. US 8,080,578 B2 Page 4
Bristow et al., “Decreased catecholamine sensitivity and Fraser et al., “Cloning, sequence analysis, and permanent expression 3-adrenergic-receptor density in failing human hearts.' N. Engl. J. of a human O-adrenergic receptor in Chinese hamster ovary cells. Med., 307(4):205-211, 1982. Evidence for independent pathways of receptor coupling to adenylate Bristow et al., “f- and 32-adrenergic receptor-mediated adenylate cyclase attenuation and activation.” J. Biol. Chem., 264:11754 cyclase stimulation in nonfailing and failing human ventricular 11761, 1989. myocardium.” Mol. Pharmacol., 35:295-303, 1988. Freeman et al., “Genetic polymorphism of the O-adrenergic receptor Bristow et al., “f- and f-adrenergic-receptor Subpopulations in is associated with increased platelet aggregation, baroreceptor sen nonfailing and failing human ventricular myocardium: coupling of sitivity, and Salt excretion in normotensive humans.” American Jour both receptor Subtypes to muscle contraction and selective f3-recep nal of Hypertension, 8(9):863-869, 1995. tor down-regulation in heart failure.” Circ. Res., 59(3):297-309, Frielle et al., “Cloning of the cDNA for the human B-adrenergic 1986. receptor.” Proc. Natl. Acad. Sci. USA, 84:7920-7924, 1987. Brodde et al., “Regional distribution of B-adrenoceptors in the human Gavin et al., “O2-adrenoceptors mediate contractile responses to heart: coexistence of functional fl- and f-adrenoceptors in both noradrenaline in the human Saphenous vein. Naunyn atria and ventricles in severe congestive cardiomyopathy, J. Schmiedeberg's Arch Pharmacol., 355:406-411, 1997. Cardiovasc. Pharmacol., 8:1235-1242, 1986. GenBank, NCBI Sequence Viewer v2.0, Accession No. U72648, Carstairs et al., “Autoradiographic visualization of beta-adrenoceptor Nov. 1998, 10 pages. subtypes in human lung.” Am. Rev. Respir: Dis., 132:541-547, 1985. Gerson et al., “Activity of the uptake-1 norepinephrine transporter as Comings et al., “Additive effect of three noradrenergic genes measured by I-123 MIBG in heart failure patients with a loss-of (ADRA2A, ADRA2C, DBH) on attention-deficit hyperactivity disor function polymorphism of the presynaptic alpha2C-adrenergic der and learning disabilities in Tourette syndrome subjects.” Clinical receptor.” Journal of Nuclear Cardiology, 10(6):583-589, 2003. Genetics, 55(3):160-172, 1999. Givertz, “Underlying causes and survival in patients with heart fail Cooper et al., “Epidemiology of a polymorphism of the B-2 ure.” N. Engl. J. Med., 342:I120-I122, 2000. adrenergic receptor gene in asthma, Am. J. Respir: Crit. Care Med., Green et al., “A polymorphism of the human fB-adrenergic receptor Abstract, 153: A254, 1996. within the fourth transmembrane domain alters ligand binding and D'Angelo et al., “Transgenic Gold overexpression induces cardiac functional properties of the receptor.” J. Biol. Chem., 268:231 16 contractile failure in mice.” Proc. Natl. Acad. Sci. U.S.A., 94:8121 23121, 1993. 8126, 1997. Green et al., “Amino-terminal polymorphisms of the human de Boer et al., “Polypharmacy in chronic heart failure: practical f3-adrenergic receptor impart distinct agonist-promoted regulatory issues regarding the use of angiotensin-converting enzyme inhibitors, properties.” Biochemistry, 33:9414-94.19, 1994. beta-blockers and other drugs.” European Society of Cardiology, Green et al., “Influence off-adrenergic receptor genotypes on signal 4(Suppl. D):D1 11-D1 16, 2002. transduction in human airway Smooth muscle cells. Am. J. Respir: Dewar et al., “The glutamine 27 f-adrenoceptor polymorphism is Cell Mol. Biol., 13:25-33, 1995. associated with elevated IgE levels in asthmatic families.”.J. Allergy Gusella, "DNA polymorphism and human disease.” Ann. Rev. Clin. Immunol., 100:261-265, 1997. Biochem., 55:831-854, 1986. Dohlman et al., “Model systems for the study of seven Hall et al., “Association of Glu 27 f-adrenoceptor polymorphism transmembrane-segment receptors.” Annu. Rev. Biochem., 60:653 with lower airway reactivity in asthmatic subjects.” The Lancet, 688, 1991. 345:1213-1214, 1995. Dorn II et al., “Mechanisms of impaired f-adrenergic receptor sig Hamid et al., “Localization off-adrenoceptor messenger RNA in naling in G-mediated cardiac hypertrophy and ventricular dysfunc human and rat lung using in situ hybridization: correlation with tion.” Molecular Pharmacology, 57:278-287, 2000. receptor autoradiography.” European Journal of Pharmacology, Dorn II et al., “O2-adrenergic receptor stimulated calcium release is 206:133-138, 1991. transduced by G-associated G-mediated activation of Heinonen et al., “Identification of a three-amino acid deletion in the phospholipase C.” Biochem., 36:6415–6423, 1997. O-adrenergic receptor that is associated with reduced basal meta Dunigan et al., “Complexity of agonist- and cyclic AMP-mediated bolic rate in obese subjects.” The Journal of Clinical Endocrinology downregulation of the human fB-adrenergic receptor: role of inter and Metabolism, 84(7):2429-2433, 1999. nalization, degradation, and mRNA destabilization.” Biochemistry, Higashi et al., “Association of a genetic variation in the f-adrenergic 41:8019-8030, 2002. receptor gene with coronary heart disease among Japanese.” Eason and Liggett, “Human O-adrenergic receptor Subtype distribu Biochem. Biophy's. Res. Comm., 232:728-730, 1997. tion: widespread and Subtype-selective expression of OC10, OC4. Himms-Hagen et al., “Effect of CL-316,243, a thermogenic and OC2 mRNA in multiple tissues.” Mol. Pharmacol. 44:70-75, 3-agonist, on energy balance and brown and white adipose tissue in 1993. rats.” Am. J. Physiol., 266:1371-1382, 1994. Eason and Liggett, "Subtype-selective desensitization of Holroyd et al., “Evidence for f3-adrenergic receptor (ADRB2) O-adrenergic receptors. Different mechanisms control short and polymorphism at amino acid 16 as a risk factor for bronchial hyper long term agonist-promoted desensitization of OC10, OC4, and responsiveness (BHR).” Am. J. Respir: Crit. Care Med. Abstract, CC2.” J. Biol. Chem., 267:25473-25479, 1992. 151:A673, 1995. Eason et al., “Simultaneous coupling of O-adrenergic receptors to Ikezuet al., “Amino acids 356-372 constitute a G-activator sequence two G-proteins with opposing effects. Subtype-selective coupling of of the O-adrenergic receptor and have a Phe Substitute in the G OC10, OC4, and OC2 adrenergic receptors to G, and G. J. Biol. protein-activator sequence motif.” FEBS Lett., 311:29-32, 1992. Chem., 267: 15795-15801, 1992. Iwai et al., “Suppressive effect of the Gly389 allele of the Eason et al., “The palmitoylated cysteine of the cytoplasmic tail of 31-adrenergic receptor gene in the occurrence of ventricular O-adrenergic receptors confers subtype-specific agonist-promoted tachycardia in dilated cardiomyopathy. Circulation Journal, downregulation.” Proc. Natl. Acad. Sci. USA,91: 11178-11182, 1994. 66:723-728, 2002. Engelhardt et al., “Progressive hypertrophy and heart failure in Jewell-Motz and Liggett, “An acidic motif within the third intracel f-adrenergic receptor transgenic mice.” Proc. Natl. Acad. Sci. U.S. lular loop of the OC2 adrenergic receptor is required for agonist A., 96:7059-7064, 1999. promoted phosphorylation and desensitization. Biochemistry, Eschenhagen et al., “Increased messenger RNA level of the inhibi 34(37): 11946-1 1953, 1995. tory G protein O. Subunit G. in human end-stage heart failure.” Jewell-Motz et al., "Agonist-mediated downregulation of G. via the Circ. Res., 70:688-696, 1992. O2-adrenergic receptor is targeted by receptor-G, interaction and is Feng et al., "Variants in the OAR adrenergic receptor gene in independent of receptor signaling and regulation. Biochemistry, psychiatric patients.” Am. J. Med. Genet., 81:405-410, 1998. 37: 15720-15725, 1998. Franz et al., “Cardiomyopathies: from genetics to the prospect of Kass, “B-receptor polymorphisms: heart failure's crystal ball.” treatment. Lancet, 358:1627-1637, 2001. Nature Medicine, 9(10): 1260-1262, 2003. US 8,080,578 B2 Page 5
Kobilka et al., “cDNA for the human B2-adrenergic receptor: a pro Moore et al., “Racial differences in the frequencies of cardiac tein with multiple membrane-spanning domains and encoded by a beta(1)-adrenergic receptor polymorphisms: analysis of c145AYG gene whose chromosomal location is shared with that of the receptor and c1 165G>C.” Hum. Mutat., 14(3):271, 1999. for platelet-derived growth factor.” Proc. Natl. Acad. Sci. USA, Morgan et al., “Yohimbine-facilitated acoustic startle reflex in 84:46-50, 1987. humans.” Psychopharmacology, 110:342-346, 1993. Kobilka et al., “Cloning, sequencing, and expression of the gene Nickerson et al., “Automated DNA diagnostics using an ELISA coding for the human platelet C2-adrenergic receptor.” Science, based oligonucleotide ligation assay.” Proc. Natl. Acad. Sci. USA, 238:650-656, 1987. 87:8923-8927, 1990. Kornher and Livak, "Mutation detection using nucleotide analogs Nyrén et al., "Solid phase DNA minisequencing by an enzymatic that alter electrophoretic mobility.” Nucl. Acids. Res., 17:7779-7784, 1989. luminometric inorganic pyrophosphate detection assay.” Anal. Krief et al., “Tissue distribution B3-adrenergic receptor mRNA in Biochem., 208:171-175, 1993. man. J. Clin. Invest., 91:344-349, 1993. Okamoto and Nishimoto, “Detection of G protein-activator regions Kumari et al., “Effect of clonidine on the human acoustic startle in M. Subtype muscarinic, cholinergic, and O-adrenergic receptors reflex.” Psychopharmacology, 123:353–360, 1996. based upon characteristics in primary structure.” J. Biol. Chem.. Kuppuswamy et al., “Single nucleotide primer extension to detect 267:8342-8346, 1992. genetic diseases: experimental application to hemophilia B (factor Orita et al., “Rapid and sensitive detection of point mutations and IX) and cystic fibrosis genes.” Proc. Natl. Acad. Sci. USA, 88: 1143 DNA polymorphisms using the polymerase chain reaction. Genom 1147, 1991. ics, 5:874-879, 1989. Kwoh et al., “Transcription-based amplification system and detection Palczewski et al., “Crystal structure of rhodopsin: a G protein of amplified human immunodeficiency virus type 1 with a bead coupled receptor.” Science, 289:739-745, 2000. based sandwich hybridization format.” Proc. Natl. Acad. Sci. USA, PCT International Preliminary Examination Report issued in Inter 86: 1173-1177, 1989. national Application No. PCT/US2003/028135, mailed Jun. 13, Landegren et al., “A ligase-mediated gene detection technique.” Sci 2006. ence, 241:1077-1080, 1988. PCT International Preliminary Examination Report issued in Inter Large et al., “Human beta-2 adrenoceptor gene polymorphisms are national Application No. PCT/US2001/012575, mailed Sep. 13, highly frequent in obesity and associate with altered adipocyte beta-2 2005. adrenoceptor function.” J. Clin. Invest., 100:3005-3013, 1997. PCT International Preliminary Report on Patentability issued in Lentes et al., “A biallelic DNA polymorphism of the human beta-2- adrenergic receptor detected by Ban I-Adrbr-2. Nucleic Acids International Application No. PCT/US2004/029838, mailed Mar. 13, Research, 16(5):2359, 1988. 2006. Li et al., “Do allelic variants in O. and O2 adrenergic receptors PCT International Preliminary Report on Patentability issued in predispose to hypertension in blacks?” Hypertension, 47: 1140-1146, International Application No. PCT/US2005/023293, mailed Jun. 19, 2006. 2007. Liggett et al., “Early and delayed consequences of C2-adrenergic PCT International Search Report issued in International Application receptor overexpression in mouse hearts.” Circ., 101: 1707-1714, No. PCT/US2005/023293, mailed May 29, 2007. 2000. PCT International Search Report issued in International Application Liggett et al., “The Ile164 f-adrenergic receptor polymorphism No. PCT/US2004/029838, mailed Oct. 14, 2005. adversely affects the outcome of congestive heart failure.” J. Clin. PCT International Search Report issued in International Application Invest., 102: 1534-1539, 1998. No. PCT/US2003/028135, mailed Nov. 22, 2005. Liggett, "B-adrenergic receptors in the failing heart: the good, the PCT International Search Report issued in International Application bad, and the unknown.” J. Clin. Invest., 107(8):947-948, 2001. No. PCT/US2001/O12575, mailed Jan. 29, 2003. Lobmeyer et al., “Synergistic polymorphisms of 3 and O PCT Invitation to Pay Additional Fees issued in International Appli adrenergic receptors and the influence on left ventricular ejection cation No. PCT/US2001/012575, mailed Nov. 11, 2002. fraction response to B-blocker therapy in heart failure.” Pertland Bianchi. "Fetal DNA in maternal plasma: emerging clinical Pharmacogenetics and Genomics, 17(4):277-282, 2007. applications.” Obstet. Gynecol. 98(3):483-490, 2001. Lomasney et al., “Expansion of the O-adrenergic receptor family: Prezant and Fischel-Ghodsian, "Trapped-oligonucleotide nucleotide cloning and characterization of a human O-adrenergic receptor Sub incorporation (TONI) assay, a simple method for screening point type, the gene for which is located on chromosome 2.” Proc. Natl. Acad. Sci. USA, 87:5094-5098, 1990. mutations.” Hum. Mutat., 1:159-164, 1992. Lowell et al., “The potential significance off adrenergic receptors.” Regan et al., “Cloning and expression of a human kidney cDNA for J. Clin. Invest., 95:923, 1995. an O-adrenergic receptor Subtype.” Proc. Natl. Acad. Sci. USA, Luttrell et al., “G-protein-coupled receptors and their regulation: 85:6301-6305, 1988. activation of the MAP kinase signaling pathway by G-protein Reihsaus et al., “Mutations in the gene encoding for thef-adrenergic coupled receptors.” Advances in Second Messenger and receptor in normal and asthmatic subjects.” Am. J. Respir: Cell Mol. Phosphoprotein Research, 31:263-277, 1997. Biol., 8:334-339, 1993. Makaritsis et al., “Role of the O-adrenergic receptor in the devel Reynisdottir et al., “Catecholamine resistance in fat cells of women opment of salt-induced hypertension.” Hypertension, 33(1): 14-17. with upper-body obesity due to decreased expression of beta 1999. adrenoceptors.” Diabetologia, 37:428-435, 1994. Martin, “Thyrotropin-releasing hormone rapidly activates the Rosin et al., “Distribution of alpha 2C-adrenergic receptor-like phosphodiester hydrolysis of polyphosphoinositides in GH pituitary immunoreactivity in the rat central nervous system. J. Comp. cells. Evidence for the role of a polyphosphoinositide-specific Neurol, 372:135-165, 1996. phospholipase C in hormone action.” J. Biol. Chem... 258: 14816 Rothwell and Stock. “A role for brown adipose tissue in diet-induced 14822, 1983. thermogenesis.” Nature, 281:31-35, 1979. McQuitty et al., “Polymorphism in the human B2 adrenergic receptor Ruffolo et al., “Pharmacologic and therapeutic applications of gene detected by Restriction Endonuclease digestion with Fnu4HI.” O-adrenoceptor subtypes.” Annu Rev Pharmacol Toxicol, 33:243 Hum. Genet., 93:225, 1994. 279, 1993. Meisel et al., “Polymorphisms of adrenergic receptors and the risk of Sakai et al., “Genetic analysis of amplified DNA with immobilized heart failure.” New England Journal of Medicine, 348(5):468-470, sequence-specific oligonucleotide probes.” Proc. Natl. Acad. Sci. 2003. USA, 86:6230-6234, 1989. Michel et al., “Functional correlates of O-adrenoceptor gene Sallinen et al., “Adrenergic O2-receptors modulate the acoustic polymorphism in the HANE study.” Nephrology, Dialysis, Trans startle reflex, prepulse inhibition, and aggression in mice.” The Jour plantation, 14(11):2657-2663, 1999. nal of Neuroscience, 18:3035-3042, 1998. US 8,080,578 B2 Page 6
Sallinen et al., “Genetic alteration of the O-adrenoceptor Subtype c in Tsujii and Bray, “Food intake of lean and obese Zucker rats following mice affects the development of behavioral despair and stress-in ventricular infusions of adrenergic agonists.” Brain Res., 587:226 duced increases in plasma corticosterone levels.” Mol. Psychiatry, 232, 1992. 4:443-452, 1999. Turki et al., “Genetic polymorphisms of the f-adrenergic receptor in Sallinen et al., “Genetic alteration of C2-adrenoceptor expression in nocturnal and nonnocturnal asthma, J. Clin. Invest., 95: 1635-1641, mice: influence on locomotor, hypothermic, and neurochemical 1995. effects of dexmedetomidine, a Subtype-nonselective O-adrenocep Turki et al., “Myocardial signaling defects and impaired cardiac toragonist.” Mol. Pharmacol. 51:36-46, 1997. function of a human f3-adrenergic receptor polymorphism expressed Saulnier-Blache et al., “Analysis of the O2-adrenergic receptor gene in transgenic mice.” Proc. Natl. Acad. Sci. USA, 93:10483-10488, 1996. promoter and its cell-type-specific activity.” Mol. Pharmacol., UgoZZoli et al., “Detection of specific alleles by using allele-specific 50: 1432-1442, 1996. primer extension followed by capture on Solid Support.” GATA, Schramm and Limbird, "Stimulation of mitogen-activated protein 9:107-112, 1992. kinase by G protein-coupled O-adrenergic receptors does not require Ungerer et al., “Altered expression of O-adrenergic receptor kinase agonist-elicited endocytosis,” J. Biol. Chem., 274:24935-24940. and f-adrenergic receptors in the failing human heart.” Circulation, 1999. 87:454-463, 1993. Schwinn et al., “The alpha 1C-adrenergic receptor: characterization van Campen et al., “Ejection fraction improvement by 3-blocker of signal transduction pathways and mammalian tissue heterogene treatment in patients with heart failure: an analysis of studies pub ity.” Mol. Pharmacol. 40.619-626, 1991. lished in the literature.” Journal of Cardiovascular Pharmacology, Serikov et al., “Reduction of Cal, restores uncoupled f-adrenergic 32(Suppl. 1):S31-S35, 1998. signaling in isolated perfused transgenic mouse hearts.” Circulation Wagoner et al., “Polymorphisms of the f-adrenergic receptor pre Research, 88:9-11, 2001. dict exercise capacity in heart failure.” American Heart Journal, Shi et al., “Distribution of alpha2-adrenoceptor mRNAs in the rat 144(5):840-846, 2002. lumbar spinal cord in normal and axotomized rats. NeuroReport, Wagoner et al., “Polymorphisms ofthef adrenergic receptor (?3AR) 10:2835-2839, 1999. affect exercise capacity in patients with heart failure' Circulation, Small and Liggett, “Identification and functional characterization of 94:8. Abstract, 1996. O-adrenoceptor polymorphisms.” TRENDS in Pharmacological Wagoner et al., “Polymorphisms of the f-adrenergic receptor deter Studies, 22(9):471-477, 2001. mine exercise capacity in patients with heart failure.” Circulation Small et al., “Identification of adrenergic receptor polymorphisms.” Research, 86:834-840, 2000. Methods in Enzymology, 343:459-475, 2002. Walker et al., “Isothermal in vitro amplification of DNA by a restric Small et al., “Polymorphic deletion of three intracellular acidic resi tion enzyme/DNA polymerase system.” Proc. Natl. Acad. Sci. USA, dues of the O-adrenergic receptor decreases G protein-coupled 89:392–396, 1992. receptor kinase-mediated phosphorylation and desensitization.” The Wartellet al., “Detecting base pair substitutions in DNA fragments by Journal of Biological Chemistry, 276(7):4917-4922, 2001. temperature-gradient gel electrophoresis.” Nucl. Acids. Res., Smith et al., “Measurement of protein using bicinchoninic acid.” 18:2699-2706, 1990. Anal. Biochem., 150:76-85, 1985. Wu et al., “The Ligation Amplification Reaction (LAR)—amplifica Snapir et al., “An insertion/deletion polymorphism in the O tion of specific DNA sequences using sequential rounds oftemplate adrenergic receptor gene is a novel genetic risk factor for acute dependent ligation.” Genomics, 4:560-569, 1989. coronary events.” Journal of the American College of Cardiology, Yang-Feng et al., “Chromosomal organization of adrenergic receptor 37(6): 1516-1522, 2001. genes.” Proc. Natl. Acad. Sci. USA, 87: 1516-1520, 1990. Sokolov, “Primer extension technique for the detection of single Yoshida et al., “Anti-obesity and anti-diabetic effects of CL 316,243, nucleotide in genomic DNA. Nucl. Acids. Res., 18:3671, 1990. a highly specific f-adrenoceptor agnoist, in yellow KK mice. Life Supplementary European Search Report issued in European Appli Sciences, 54:491-498, 1994. cation No. EP 05789 151, mailed Sep. 5, 2008. Yussman et al., “Mitochondrial death protein Nix is induced in car Supplementary European Search Report issued in European Appli diac hypertrophy and triggers apoptotic cardiomyopathy.” Nature cation No. 048161814, mailed Oct. 11, 2006. Medicine, 8(7):725-730, 2002. Supplementary Partial European Search Report issued in European Altman et al., "Abnormal regulation of the sympathetic nervous Application No. EP 03794666.2, mailed Jul. 17, 2006. system in O-adrenergic receptor knockout mice.” Mol. Pharmacol., Syvänen et al., “A primer-guided nucleotide incorporation assay in 56:154-161, 1999. the genotyping of apolipoprotein E.” Genomics, 8:684-692, 1990. Arner and Hoffstedt, Adrenoceptor genes in human obesity,” Jour Syvänen et al., “Identification of individuals by analysis of biallelic nal of Internal Medicine, 245:667-672, 1999. DNA markers, using PCR and Solid-phase minisequencing.” Amer: J. Baron and Siegel, “p-'IIIodoclonidine, a novel radiolabeled Hum. Genet., 52:46-59, 1993. agonist for studying central O-adrenergic receptors.” Mol. Tan et al., “Association between 32-adrenoceptor polymorphism and Pharmacol. 38:348-356, 1990. Susceptibility to bronchodilator desensitisation in moderately severe Cotton, "Current methods of mutation detection.” Mutation stable asthmatics. Lancet, 350:995-999, 1997. Research, 285:125-144, 1993. Tan et al., “f-adrenoceptor polymorphism determines Susceptibility Dao et al., “Expression of altered O-adrenergic phenotypic traits in to bronchodilator desensitisation in asthmatics.” Am. J. Respir: Crit. normotensive humans at genetic risk of hereditary (essential) hyper Care Med., Abstract, 155:A208, 1997. tension.” J Hypertens., 16(6);779-792, 1998. Tanila et al., “Role of O-adrenoceptor Subtype in spatial working de Chasseval and de Villartay, “High level transient gene expression memory as revealed by mice with targeted disruption of the O in human lymphoid cells by SV40 large T antigen boost.” Nucleic adrenoceptor gene.” European Journal of Neuroscience, 11:599-603, Acids Res., 2002):245-250, 1992. 1999. Eason and Liggett, "Chimeric mutagenesis of putative G-protein Tepe et al., “Altering the receptor-effector ratio by transgenic coupling domains of the O-adrenergic receptor. Localization of two overexpression of type V adenylyl cyclase: enhanced basal catalytic redundant and fully competent G, coupling domains.”J Biol Chem. activity and function without increased cardiomyocyte f-adrenergic 271 (22): 12826-12832, 1996. signalling.” Biochemistry, 38:16706-16713, 1999. Eason et al., “Contribution of ligand structure to activation of Tesson et al., "Characterization of a unique genetic variant in the O2-adrenergic receptor Subtype coupling to G. Mol. Pharmacol., 31-adrenoceptor gene and evaluation of its role in idiopathic dilated 45:696-702, 1994. cardiomyopathy.” Journal of Molecular and Cellular Cardiology, Eason et al., “Four consecutive serines in the third intracellular loop 31:1025-1032, 1999. are the sites for 3-adrenergic receptor kinase-mediated phosphoryla Trayhurn and Mercer, “Brown adipose tissue thermogenesis in obese tion and desensitization of the O-adrenergic receptor.” The Journal animals.” Biochem. Soc. Trans., 14:236-239, 1986. of Biological Chemistry, 270(9):4681-4688, 1995. US 8,080,578 B2 Page 7
Exton, “Regulation of phosphoinositide phospholipases by hor Luttrell et al., “Regulation of mitogen-activated protein kinase path mones, neurotransmitters, and other agonists linked to G proteins.” ways by catecholamine receptors.” Adv. Pharmacol., 42:466-470, Annu Rev Pharmacol Toxicol. 36:481-509, 1996. 1998. Goldstein et al., “Sympathetic reactivity during a yohimbine chal MacMillan et al., “Central hypotensive effects of the O-adrenergic lenge test in essential hypertension.” Hypertension., 18(5 Sup receptor subtype.” Science, 273:801-805, 1996. pl):III40-III48, 1991. MacMillan et al., “O2-adrenergic receptor Subtypes: Subtle mutation Goodman and Gilman's The Pharmacological Basis of Therapeutics, of the O-adrenergic receptor in vivo by gene targeting strategies Pergamon Press, 1990, New York, pp. 89-90. reveals the role of this subtype in multiple physiological settings.” Gratze et al. "3-2 adrenergic receptor variations affect resting blood Recent Prog Horm Res., 53:25-42, 1998. pressure and agnoist-induced vasodilation in young adult Cauca Makaritsis et al., “Sympathoinhibitory function of the O sians.” Hypertension, 33: 1425-1430, 1999. adrenergic receptor subtype.” Hypertension, 34(3):403-407. 1999. Guyer et al., “Cloning, sequencing, and expression of the gene encod Martin-Guerrero et al., “The N251K functional polymorphism in the ing the porcine O-adrenergic receptor.” The Journal of Biological O-adrenoceptor gene is not associated with depression: a study in Chemistry, 265(28): 17307-17317, 1990. suicide completers.” Pyschopharmacology, 184:82-86, 2006. Handy et al., “Diversetissue expression of rat O-adrenergic receptor Meirhaeghe et al., “f-adrenoceptor gene polymorphism, body genes.” Hypertension, 21: 861-865, 1993. weight, and physical activity.” Lancet, 353:896, 1999. Hausdorff et al., “Phosphorylation sites on two domains of the Munroe and Caufield, “Genetics of hypertension.” Current Opinion 32-adrenergic receptor are involved in distinct pathways of receptor in Genetics & Development, 10:325-329, 2000. desensitization.” The Journal of Biological Chemistry, 264:12657 Newton, “Primers,” in PCR Essential Data, Chapter 6, pp. 49-56, 12665, 1989. 1995. Hein et al., “Gene substitution knockout to delineate the role of Onions et al., “Genetic markers at the leptin (OB) locus are not O-adrenoceptor Subtypes in mediating central effects of significantly linked to hypertension in African Americans. Hyper catecholamines and imidazolines.” Annals New York Academy of tension, 31(6):1230-1234, 1998. Sciences, 881:265-271, 1999. Onorato et al., “Role of acidic amino acids in peptide substrates of the Hellström et al., “The different effects of a Gln27Glu B-adrenocep 3-adrenergic receptor kinase and rhodopsin kinase. Biochemistry, tor gene polymorphism on obesity in males and in females.” Journal 30:51 18-5125, 1991. of Internal Medicine, 245:253-259, 1999. Pitcher et al., “G protein-coupled receptor kinases.” Annu. Rev. Ho et al., “Structural polymorphism in the O2 adrenoceptor: no Biochem., 67:653-692, 1998. association with Schizophrenia or clozapine responsiveness. Am. J. Pollin et al., “The Glu'/Glu' mutation in the O2-adrenergic receptor Med. Genet., 81(6):510, Abstract No. 93, 1998. is associated with increased resting metabolic rate (RMR) in a Cau Holmes et al., “Guanabenz. A review of its pharmacodynamic prop casian cohort.” Obesity Research, 8:Supp 859, Abstract No. PE4. erties and therapeutic efficacy in hypertension.” Drugs, 26(3):212 2000. 229, 1983. Reihsaus et al., “Mutations in the gene encoding for the beta Ishiyama-Shigemoto et al., "Association of polymorphisms in the 2-adrenergic receptor in normal and asthmatic Subjects.” Am. J. 32-adrenergic receptor gene with obesity, hypertriglyceridaemia, Respir: Cell Mol. Biol., 8:334-339, 1993. and diabetes mellitus.” Diabetologia, 42:98-101, 1999. Robinson and Hudson, “Andrenoceptor pharmacology, located Jewell-Motz and Liggett, "G protein-coupled receptor kinase speci online at http://www.tocris.com/adrenoceptor.htm, 1998. (Accessed ficity for phosphorylation and desensitization of the O-adrenergic on Mar. 19, 2001). receptor subtypes.” The Journal of Biological Chemistry, Rohrer and Kobilka, “G protein-coupled receptors: functional and 271 (30): 18082-18087, 1996. mechanistic insights through altered gene expression.” Physiological Jewell-Motz et al., “O/O-adrenergic receptor third loop chimera Reviews, 78(1):35-52, 1998. show that agonist interaction with receptor Subtype backbone estab Sakane et al., “f-adrenoceptor gene polymorphism and obesity.” lishes G protein-coupled receptor kinase phosphorylation.” The Jour Lancet, 353:1976, 1999. nal of Biological Chemistry, 275(37): 28989-28993, 2000. Salonen et al., “An insertion/deletion polymorphism in the alpha-2b Kotanko et al., “Essential hypertension in African Caribbean associ adrenergic receptor gene is a novel genetic risk factor for acute ates with a variant of the f-adrenoceptor.” Hypertension, 30:773 coronary events.” Circulation, 102(18): Supp 859, Abstract No. 4125. 776, 1997. 2000. Kurose and Lefkowitz. “Differential desensitization and Shenker et al., “A constitutively activating mutation of the luteinizing phosphorylation of three cloned and transfected O-adrenergic recep hormone receptor in familial male precocious puberty.” Nature, tor subtypes,” J. Biol. Chem., 269:10093-10099, 1994. 365(6447):652-654, 1993. Lakhlani et al., "Substitution of a mutant O-adrenergic receptor via Small et al., “An ASn to Lys polymorphism in the third intracellular "hit and run' gene targeting reveals the role of this subtype in Seda loop of the human O-adrenergic receptor imparts enhanced tive, analgesic, and anesthetic-sparing responses in vivo. Proc Natl agonist-promoted G coupling.” The Journal of Biological Chemistry, AcadSci USA., 94(18):9950-9955, 1997. 275(49):385 18-38523, 2000. Liggett et al., “Adrenergic receptor-coupled adenylyl cylase systems: Spiegel, “Defects in G protein-coupled signal transduction in human regulation of receptor function by phosphorylation, sequestration and disease.” Annu Rev Physio., 58:143-170, 1996. downregulation.” In Regulation of Cellular Signal Transduction Svetkey et al., "Association of hypertension with f3- and Co Pathways by Desensitization and Amplification, pp. 71-97, 1993. adrenergic receptor genotype.” Hypertension, 27(6): 1210-1215, Liggett et al., “Sites in the third intracellular loop of the O 1996. adrenergic receptor confer short term agnoist-promoted desensitiza Svetkey et al., “Preliminary evidence of linkage of saltsensitivity in tion.” The Journal of Biological Chemistry, 267(7):4740-4746, 1992. black Americans at the f-adrenergic receptor locus. Hypertension, Liggett, "Pharmacogenetics of relevant targets in asthma. Clin Exp 29:918-922, 1997. Allergy, 28 Suppl 1:77-79; discussion 80-81, 1998. Tavares et al., “Localization of O2- and O-adrenergic receptor Limbird, “Receptors linked to inhibition of adneylate cyclase: addi subtypes in brain.” Hypertension, 27:449-455, 1996. tional signaling mechanisms.” FASEB.J., 2:2686-2695, 1988. Timmermann et al., “f-2 adrenoceptor genetic variation is associated Link et al., "Cardiovascular regulation in mice lacking O with genetic predisposition to essential hypertension: the Bergen adrenergic receptor subtypes b and c.” Science, 273:803-805, 1996. blood pressure study.” Kidney International, 53:1455-1460, 1998. Lockette et al., “O2-adrenergic receptor gene polymorphism and hypertension in blacks.” Am J Hypertens., 8(4 Pt 1):390-394, 1995. * cited by examiner U.S. Patent Dec. 20, 2011 Sheet 1 of 29 US 8,080,578 B2
Bucindolol 2-(3-(1-(1H-indol-3-yl)-2-methylpropan-2-ylamino)-2-hydroxypropoxy)benzonitrile
Atenolo 2-(4-(2-hydroxy-3-(isopropylamino)propoxy)phenyl)acetamide
OH
Hco Metoprolol 1-(isopropylamino)-3-(4-(2-methoxyethyl)phenoxy)propan-2-ol
OH O N-1N Carvedilol OCH 1-carbazolyl-(4)-oxy-3-(2-methoxyphenoxyethyl) amino)-2-propanol F.G. 1 U.S. Patent Dec. 20, 2011 Sheet 2 of 29 US 8,080,578 B2
(+7-0)sa?uedoudo?6olooeuue?,‘punoduuoo Z“?INH
U.S. Patent Dec. 20, 2011 Sheet 3 of 29 US 8,080,578 B2
600 : f 1 -Arg389 A. G C 9 a. S 400 s t E B-Gly389 y 200
O ------O -9 -8 -7 -6 -5 -4. log bucindolol, M.
FIG. 3 U.S. Patent Dec. 20, 2011 Sheet 4 of 29 US 8,080,578 B2
B-Gly389 mice 25 untreated propranolol
200
150 1OO III O GS Gi GRK2 AC5 P-PLN T-PLN SERCA
B1-Arg389 mice
250 untreated propranolol
2OO
150
1OO
50
O GS G i GRK2 AC5 P-PLN T-PLN SERCA
FIG. 4 U.S. Patent US 8,080,578 B2
-
S"?INH
| U.S. Patent US 8,080,578 B2
099|09G||0#7063
04.??.
090
- O 8'O 4. GO All CeCIOdd eAAunS U.S. Patent Dec. 20, 2011 Sheet 7 Of 29 US 8,080,578 B2
–!
--º--~------
- 9'O O KIQeq0id uIOdpu U.S. Patent
-00|| |0E' 093| |ÇD -09-) -001 ? pn 2 |© U.S. Patent Dec. 20, 2011 Sheet 9 Of 29 US 8,080,578 B2
6(5)IH 9°00
?S?:puZ VZ
U.S. Patent Dec. 20, 2011 Sheet 11 of 29 US 8,080,578 B2
II‘5)IH U.S. Patent Dec. 20, 2011 Sheet 12 of 29 US 8,080,578 B2
rt > CD r ism O) & O O)C S. S S 9) 9 CC o S Ol O on 9
co------rif c d co co v c toco S v- w w w - C m s 2. (D is - firm C) C. O) s . n s 9 E S O aC * C O o ------e < venp viast snvs vale o co st on o uu/Nu) e3O.
U.S. Patent Dec. 20, 2011 Sheet 14 of 29 US 8,080,578 B2
?I(5)IH
U.S. Patent Dec. 20, 2011 Sheet 18 Of 29 US 8,080,578 B2
·ENolure?sºspueadá ul,s?uouuche(W?s+·LSaa
8I"?INH U.S. Patent Dec. 20, 2011 Sheet 19 Of 29 US 8,080,578 B2
U.S. Patent Dec. 20, 2011 Sheet 21 of 29 US 8,080,578 B2
IZ* ?ÌNH
U.S. Patent Dec. 20, 2011 Sheet 22 of 29 US 8,080,578 B2
@@@ ©ïae. “suopelndo, @:.
· *, *…
ZZ"?INH
U.S. Patent Dec. 20, 2011 Sheet 26 of 29 US 8,080,578 B2
Xs
unnu) aero
|.(:)tênialfoi
U.S. Patent Dec. 20, 2011 Sheet 28 Of 29 US 8,080,578 B2
IIuásøOIÁpngS 8Z"?INH
IISSBIO
U.S. Patent Dec. 20, 2011 Sheet 29 Of 29 US 8,080,578 B2
US 8,080,578 B2 1. 2 METHODS FOR TREATMENT WITH prises only 12% of the American HF population (Taylor et al., BUCNDOLOL BASED ON GENETIC 2004). Clearly, there is a continued need to develop the next TARGETING generation of HF drugs. While Bagonists are used for treating acute deterioration This application is a divisional of U.S. patent application of patients with failing ventricular function, prolonged expo Ser. No. 1 1/226.908 filed Sep. 14, 2005, which claims priority sure of the heart from administered agonists, or the elevated to U.S. Provisional Patent Application No. 60/609.689 filed catecholamine agonists produced by the body, leads to wors on Sep. 14, 2004 and U.S. Provisional Patent Application No. ening heart failure. In contrast, 3-adrenergic receptor antago 60/610,706 filed on Sep. 17, 2004, all of which are hereby nists (termed B-blockers) have emerged as a major treatment incorporated by reference in their entirety. 10 modality in chronic heart failure. This invention was made with government Support under In the early 1990s, a group of U.S. heart failure investiga grants HL052318, HL07071609, ES06096, HL07 1118, and tors working with B-blocking agents in heart failure decided HL48013 awarded by the National Institutes of Health. The that a mortality trial was required in order to validate this government has certain rights in the invention. still-controversial therapy. A group of U.S. drafted a protocol 15 and grant application that was Subsequently approved for BACKGROUND OF THE INVENTION funding by the VA cooperative Clinical Studies Program and the NHLBI. The approved protocol did not specify a drug, but 1. Field of the Invention rather provided that an optimal B-blocker would be selected The present invention relates to pharmacogenetics and car based on potential for Success and strength of Phase II data. diology. More specifically, the present invention relates to The drugs that were considered were carvedilol, metoprolol methods for individualized heart failure therapy with bucin tartrate, metoprolol succinate CR/XL, and bucindolol. Meto dolol based on a patient's genotype of polymorphisms in prolol tartrate was rejected because of less than promising adrenergic receptor genes, including the B-adrenergic recep effects on mortality from the MDC Trial (Waagstein et al., tor (BAR) gene and the C2-adrenergic receptor (CAR) 1993); metoprolol succinate CR/XL was not selected because gene. 25 of a lack of efficacy and tolerability data in heart failure; and 2. Description of Related Art carvedilol was not selected in part because of poor tolerability According to the American Heart Association (AHA), in advanced heart failure (Krum et al. 1995). Bucindolol was about 62 million Americans have some form of cardiovascu the unanimous choice of the Selection Committee, based on lar disease, which can include high blood pressure, coronary its excellent tolerability (Eichhornet al., 1997: Gilbert et al., heart disease (heart attack and chest pain), Stroke, birth 30 1990; Bristow et al., 1994; Pollock et al., 1990), efficacy defects of the heart and blood vessels, and congestive heart (Gilbert et al., 1990; Bristow et al., 1994; Pollocket al., 1990; failure, and close to a million die from such conditions every Eichhorn et al., 1990), and level of interest by its sponsor. year. The annual report of the AHA further states that cardio Bucindolol thus became the subject of the Beta Blocker vascular disease kills more Americans than the next seven Evaluation of Survival Trial (“BEST), the first mortality trial causes of death combined, including cancer. Surprisingly, 35 planned and initiated in HF. slightly more females, overall, than males have cardiovascu The BEST trial began in 1995, and ended in 1999. After lar disease. Heart disease accounted for 40% of all deaths in BEST was initiated three other mortality trials were planned the U.S. in 1999. Despite recent treatment advances, mortal and initiated, MERIT-HF with metoprolol succinate CR/XL ity from heart failure is approximately 50% within 5 years. (MERIT-HF Study Group, 1999), CIBIS-II with bisoprolol In the United States alone there are approximately six 40 (CIBIS-II Investigators, 1999), and COPERNICUS with million people, about 1.5% of the population, with chronic carvedilol (Packeret al., 2002). Due to the more rapid and less heart failure (“HF), and 550,000 new patients are diagnosed restrictive enrollment of these trials, CIBIS-II and MERIT each year. Medical therapy has made progress in treating HF, HF were completed before BEST, and both these trials had but morbidity and mortality remain high (Mann et al., 2005). positive results. The current standard of care in HF involves the use of inhibi 45 The BEST Trial was terminated prematurely in 1999, prior tors (ACE inhibitors, ARBs, and/or aldosterone receptor to completion, due in part to a loss of equipoise by investiga antagonists) of the renin-angiotensin-aldosterone system tors, and an accelerated drop-in rate to open label B-blockers (RAAS), and n-blockers, which competitively inhibit B-adr based on the knowledge of the other two positive trials (BEST energic receptors on cardiac myocytes. B-blockers are effec Trial Investigators, 2001; Domanski et al., 2003). The spon tive in mortality reduction and are considered the most effec 50 sor elected not to proceed with the commercial development tive HF drug class overall, but still work in only 50-60% of ofbucindolol based on the results known at the time the Trial treated patients. Moreover, the U.S. patient clinical data on was stopped. While BEST investigators observed a benefit in the efficacy of approved f-blockers in mortality trials is less Class III, non-African-Americans, that was similar to the impressive, with published data from the only large, inten positive results reported six months earlier in CIBIS II and tion-to-treat mortality trial showing an increase in mortality 55 MERIT-HF, the investigators observed poor results in Class in U.S. patients vs. placebo. IV and African-American patients. Moreover, BEST did not Accordingly, there is a substantial need for improved HF meet its primary endpoint of all-cause mortality (reduction of therapies that have higher efficacy and response rates, are 10%, B=0.10) when the trial was stopped (BEST Trial Inves better tolerated, and are better suited to subpopulations with tigators, 2001). The investigators postulated that the differ special needs, such as diabetics. However, development of 60 ences between the results of other (3-blockers and bucindolol new agents against this therapeutic background has proved might be attributable to the “unique pharmacological proper extremely challenging. Since 2001, of 13 Phase III trials in ties of bucindolol (BEST Trial Investigators, 2001), which HF only three have been positive. Two of these positive trials highlights the perceived distinctions among the chemical and were with an ARB (candesartan) (McMurray et al., 2003: functional properties of this diverse class of compounds. Granger et al., 2003). The third positive trial, the A-HeFT 65 Moreover, even though most B-blocker trials in heart fail Trial with BiDil (a combination of isosorbide dinitrate and ure have shown group beneficial effects, there is substantial hydralazine) was in a Subset (African-Americans) that com interindividual variability in outcome that is not explained by US 8,080,578 B2 3 4 baseline clinical characteristics (CIBIS-II Investigators, tein and another at nucleotide positions 964-975 of the CAR 1999). Interindividual variability in the response to pharma gene that corresponds to amino acid positions 322-325 in the cologic therapy is recognized with virtually all drugs. In encoded protein. circumstances Such as the treatment of chronic heart failure The invention is based on the determination by the inven with B-blockers—where morbidity and mortality are high, tors that being homozygous in the BAR gene to encode an the titration algorithm is complex, the interindividual Vari arginine at position 389 in the gene product provides the ability is Substantial, and additional treatment options exist— patient with a physiology that is amenable to treatment with assessing the likelihood of a favorable (or adverse) long-term bucindolol. In addition, the invention is based on the deter response to drug therapy can have a significant impact on mination that a deletion in the CAR gene that leads to a decision making. The approximately 50% 5-year mortality of 10 deletion of amino acids 322-325 in the gene product is detri mental to treatment of later stages of heart failure with bucin patients with heart failure has prompted intense study of dolol. The term “treatment” will be understood to refer to treatment options and has lead to multidrug regimens typi therapy with respect to a patient diagnosed with a cardiovas cally including a B-blocker, an angiotensin converting cular disease or with symptoms of a cardiovascular, as enzyme inhibitor (orangiotensin receptor antagonist), diuret 15 opposed to preventative measures. ics, and digoxin. B-blocker therapy is initiated in relatively It is generally understood that polymorphisms occur in the stable patients, at low doses (i.e., about 10 mg), and slowly context of genes; however, in the case of polymorphisms that increased over a period of months to either a target dose, or a affect the encoded gene product, an alteration in that gene dose which is tolerated. Dosage adjustments of other drugs, product may also be referred to as a polymorphism. or initiation of additional drugs is not uncommon during the According to the invention, methods include assessing up-titration period. Thus the treatment of heart failure with whether to prescribe or administer bucindolol to a patient (3-blockers must be individualized. Indeed the statement with cardiovascular disease comprising obtaining informa “dosage must be individualized and closely monitored” is tion from the patient regarding his/her polymorphisms in found in the prescribing information for the two B-blocker adrenergic receptor genes and/or their encoded gene products preparations approved for treating heart failure in the U.S. 25 that affect a response to bucindolol. Furthermore, studies in animal models and humans suggest Therefore, the present invention is concerned with obtain that f-blocker-promoted reversal of the cellular and global ing the information regarding polymorphisms in the BAR remodeling of the failing heart may require months of stable and/or CAR proteins directly or as deduced by determining therapy (Lowes et al., 2002). Substantial variability in the nucleotide sequence at position 1165 on the BAR gene responses to (3-blockers has been noted, including left ven 30 and/or positions 964-975 on the CAR gene, and prescribing or administering bucindolol based on the obtained informa tricular ejection fraction (LVEF) changes (van Campen et al., tion. It will be understood that cognate nucleic acids for the 1998), exercise tolerance (Bolger, 2003) and survival (Packe f3AR or CAR protein include the mRNA transcript encod et al., 2001). Nevertheless, based on the preponderance of ing the protein, both strands of any cDNA generated from the data, B-blocker therapy should be considered for most 35 mRNA transcript, and both strands of the genomic DNA for patients with chronic heart failure, assuming no contraindi the BAR or CAR gene. cations such as Volume overload, requirement for inotropic Knowledge about which polymorphism a cardiovascular infusions, bradycardia, hemodynamic instability, and disease patient has at position 389 of the BAR and/or posi asthma. tions 322-325 of CAR provides the basis for assessing Consequently, not only were there perceived differences 40 whether to administer or prescribe bucindolol to the patient. among the various B-blockers—particularly bucindolol as The present invention further identifies patients with heart compared to other B-blockers—but also variability had been failure that will positively respond to treatment using observed among patients in their abilities to respond favor B-blockers, specifically bucindolol. able to a particular B-blocker therapy. Evidence for the thera The present invention also provides devices and composi peutic value of bucindolol is needed, particularly evidence 45 tions for the delivery of B-blockers, specifically bucindolol, to that explains these interindividual differences. an individual in need of Such therapy. The method of the present invention comprises determin SUMMARY OF THE INVENTION ing the genotype for a heart failure patient at the individuals f3AR gene, wherein the patient is likely to exhibit a positive The present invention provides methods for individualized 50 response to a standard dose ofbucindolol if the patient is not cardiovascular disease therapy based on the identification of a carrier of BGly389 (that is, having one or two Gly389 polymorphisms in adrenergic receptors that affect an indi alleles). In an embodiment, bucindolol is prescribed for a vidual’s response to bucindolol. In certain embodiments, it heart failure patient who is homozygous for BArg389. The concerns individualized therapy for heart failure. method of the present invention further contemplates pre In certain embodiments, there are methods for evaluating 55 scribing or administering a standard dose of bucindolol to a bucindolol treatment for a patient comprising obtaining patient in need of Such therapy based on knowing that the sequence information regarding at least one polymorphism in patient is "homozygous BArg389, meaning both BAR an adrenergic receptor gene of the patient, wherein the infor genes of the patient encode an arginine at position 389 in the mation is predictive of bucindolol efficacy in the patient. The gene products. Methods of the invention involve prescribing sequence information may be nucleic acid sequence informa 60 or administering bucindolol to patients who are homozygous tion and/or amino acid sequence information. In particular fArg389 and this is regardless of how it is determined that embodiments, the adrenergic receptor gene is BAR or the patient has that genotype. CAR. In some cases, sequence information about a poly In further embodiments, the method of the present inven morphism in both genes is obtained. tion comprises determining the genotype for a heart failure Moreover, the polymorphisms include one at nucleotide 65 patient at the individual’s CAR gene, wherein the patient is position 1165 in the B-adrenergic receptor (BAR) gene that likely to exhibit a positive response to a standard dose of corresponds to amino acid position 389 in the encoded pro bucindolol if the patient is not a carrier of C. Del322-325. In US 8,080,578 B2 5 6 certain embodiments, bucindolol is prescribed for a heart Other aspects of the invention include methods for treating failure patient who is homozygous wildtype for Catamino a patient with a heart condition may comprise administering acid positions 322-325 (i.e., the amino acids are not deleted). or prescribing to the patient an effective amount of bucin The method of the present invention further contemplates dolol, wherein the patient does not have detectable levels of a prescribing or administering a standard dose ofbucindolol to 5 BAR protein with a glycine at position 389 or wherein the a patient in need of Such therapy based on knowing that the patient is homozygous for a cytosine at position 1165 in the patient is "homozygous wildtype CAR. meaning the nucleotide coding sequence of both BAR alleles. In either patient does not have a deletion in the CAR gene sequence case a doctor or other medical practitioner may prescribe or that encodes amino acids 322-325 in CAR. Methods of the administer bucindolol if they are aware that bucindolol is an invention involve prescribing or administering bucindolol to 10 appropriate medication for that patient by virtue of that patients who are homozygous wildtype for the deletion (not a patient having the Arg389/Arg389 polymorphism in the deletion carrier) and this is regardless of how it is determined BAR gene. that the patient has that genotype. Alternatively or additionally, methods for treating a patient It is contemplated that in certain situations, a patient may with a heart condition may comprise administering or pre be genotyped for one of these polymorphisms and then a 15 scribing to the patient an effective amount of bucindolol, Subsequent determination is done with respect to the other wherein the patient does not have detectable levels of a BAR polymorphism; in this scenario, two different samples are protein with a deletion of amino acids 322-325 or wherein the evaluated. Alternatively, a single sample may be obtained and patient is homozygous for the presence of nucleotides 964 evaluated for two separate polymorphisms. In another 975 (“nondeletion') in the coding sequence of both CAR embodiment, bucindolol is prescribed for a heart failure 20 alleles. In either case a doctor or other medical practitioner patient who has the diplotype of homozygous f Arg389 and may prescribe or administer bucindolol if they are aware that homozygous wild type CAR. The method of the present bucindolol is an appropriate medication for that patient by invention further contemplates prescribing or administering a virtue of that patient not being a carrier of the Del322-325 standard dose of bucindolol to a patient in need of such polymorphism in the CAR gene, that is, not being heterozy therapy based on knowing that the patient is homozygous for 25 gous or homozygous for the deletion. BArg389 or for wild type CAR, or the diplotypic combi Additional methods include evaluating whether a heart nation. failure patient will respond positively to a bucindolol com The method of the present invention also comprises deter prising: a) obtaining information indicatingi) the presence of mining whether individuals having similar pathophysiologi a polymorphism at the coding position 1165 in the coding cal states, such as but not limited to, dilated cardiomyopathy, 30 sequence of one or both f3AR genes of the patient or ii) the ischemic cardiomyopathy, ischemic heart disease (angina, presence of a polymorphism at the amino acid at position 389 myocardial infarction), pheochromocytoma, migraines, car of the BAR protein; and b) prescribing or administering diac arrhythmias, hypertension and various anxiety disorders bucindolol. are likely to positively respond to a standard dose of bucin Moreover, other methods covered by the invention involve dolol based on whether the individual is homozygous for 35 treating a patient with bucindolol comprising: a) obtaining Arg389 at the individual’s BAR gene (and not a carrier of the information indicating i) the presence of a polymorphism at 389Gly) and/or whether the individual is homozygous wild the coding position 1165 in the coding sequence of one or type for CAR gene and not a carrier of ODel322-325. both BAR alleles of the patient or ii) the presence of a In certain embodiments, the invention concerns methods polymorphism at the amino acid at position 389 of the BAR for evaluating bucindolol treatment for a patient comprising 40 protein; and b) either prescribing bucindolol therapy for the knowing either (i) the sequence at nucleotideposition 1165 of patient wherein the patient's genotype indicates the patient is one or both coding sequences of the patient’s faR genes or homozygous Arg389 in the faR protein or not prescribing (ii) the amino acid at position 389 of the patient’s BAR bucindolol for the patient wherein the patients genotype proteins, wherein the individual is being considered for treat indicates the patient is not homozygous Arg389 in the AR ment with bucindolol. 45 protein. In further embodiments, the invention concerns methods It is further contemplated that the invention concerns the for evaluating bucindolol treatment for a patient comprising use ofbucindolol in the manufacture of a medicament for the knowing whether there is a deletion either (i) in the sequence treatment of a heart condition in patients with the Arg389/ at nucleotidepositions 964-975 of one or both of the patients Arg389 polymorphism in their BAR genes. The embodi CAR alleles or (ii) in the amino acids at positions 322-325 50 ments discussed with respect to methods may be imple of the patient’s CAR proteins, wherein the individual is mented in use of bucindolol in the manufacture of a being considered for treatment with bucindolol. medicament. It is contemplated that not all of the patient’s proteins will Also, the present invention concerns obtaining a biological be evaluated in any embodiment of the invention but that a sample from a patient who is being considered for treatment sample will be obtained and some of the proteins in the 55 with bucindolol and evaluating it for the Arg389 polymor sample will be evaluated for their protein sequence. phism by determining either (i) the sequence at nucleotide It is also contemplated that the term “knowing is used position 1165 of one or both coding sequences of the patients according to its ordinary and plain meaning to refer to having BAR genes or (ii) the amino acid at position 389 of the the specified information. It is contemplated that typically a patient’s BAR proteins. It is contemplated that if BAR pro medical practitioner will be evaluating whether to prescribe 60 teins are evaluated, one might look for whether a sample or administer a patient bucindolol and in making that evalu contains any faR proteins with a glycine at 389. ation the practitioner will order one or more tests regarding Further methods include evaluating whether a heart failure one or both of the patient’s BAR alleles or their encoded patient will respond positively to a bucindolol comprising: a) proteins and/or regarding one or both of the patient’s CAR obtaining information indicating whether i) the nucleotide alleles or their encoded proteins. In the context of the poly- 65 sequence at positions 964-975 has been deleted in one or both morphisms discussed herein, the terms “allele' and “gene’ of the patient's CAR alleles or ii) the amino acid sequence are used interchangeably. at positions 322-325 has been deleted in the patient’s CAR US 8,080,578 B2 7 8 proteins; and b) prescribing or administering bucindolol. It is option. It is contemplated that, for example, a laboratory contemplated that if CAR proteins are evaluated, one might conducts the test to determine that patient's genotype such its look for whether a sample contains any CAR proteins with personnel also know the appropriate information. They may the relevant deletion. report back to the practitioner with the specific result of the Moreover, other methods covered by the invention involve test performed or the laboratory may simply report that bucin treating a patient with bucindolol comprising: a) obtaining dolol is appropriate drug based on the laboratory results. information indicating whether i) the nucleotide sequence at In further embodiments, the patient's genotype at nucle positions 964-975 has been deleted in one or both of the otide position 1165 of the coding sequence of one or both patient’s CAR alleles or ii) the amino acid sequence at f3AR alleles is known. In the context of the present invention, positions 322-325 has been deleted in the patients a AR 10 whether position 1165 of the coding sequence contains a proteins; and b) either prescribing bucindolol therapy for the guanine or cystosine in one or both alleles is significant. This patient wherein the patient's genotype indicates the patient is indicates whatamino acid can be found at position 389 of the homozygous wildtype in the CAR alleles or not prescribing BAR protein sequence. A cytosine at position 1165 in the bucindolol for the patient wherein the patient’s genotype coding sequence encodes an arginine, while a guanine in the indicates the patient is not homozygous wildtype in the 15 coding sequence encodes a glycine. In particular embodi CAR protein. ments, the sequences at position 1165 in both BAR alleles of It is further contemplated that the invention concerns the the patient are known. The result may be a guanine in both use ofbucindolol in the manufacture of a medicament for the alleles, a cytosine in bothalleles, oraguanine in one allele and treatment of a heart condition in patients with the homozy a cytosine in the other allele. gous wildtype 322-325 polymorphism in their CAR alleles. In certain embodiments, the patient's genotype at nucle The embodiments discussed with respect to methods may be otidepositions 964-975 of the coding sequence of one or both implemented in use of bucindolol in the manufacture of a CAR alleles is known. In the context of the present inven medicament. tion, whether there is a deletion in one or both alleles of the Also, the present invention concerns obtaining a biological CAR gene is significant. This indicates whether there is a sample from a patient who is being considered for treatment 25 deletion amino acid of amino acids 322-325 (amino acids with bucindolol and evaluating it for the Arg389 polymor 322, 323, 324, and 325) of the CAR protein sequence. In phism in the BAR protein and/or the Del322-325 polymor particular embodiments, whether there is a deletion of the phism in the CAR protein by determining (i) the sequence at nucleotide sequence corresponding to positions 964-975 in nucleotide position 1165 of one or both coding sequences of both BAR alleles of the patient is known. the patient's BAR alleles; (ii) the amino acid at position 389 30 Those of skill in the art readily understand that the coding in the patient’s BAR proteins; iii) whether there is a deletion sequence of a gene refers to the Strand of the gene that is used in nucleotides 964-975 in the coding sequence of one or both for transcription of messenger RNA. The sequence of the CAR alleles; and/or iv) whether there is a deletion of amino coding sequence is complementary to the sequence of the acids 322-325 in the patient’s CAR proteins. transcribed transcript. Because of the complementary nature To achieve these methods, a doctor, medical practitioner, or 35 of sequences between a coding sequence and a noncoding their staff may obtain a biological sample for evaluation. The sequence, the sequence of any coding sequence can be deter sample may be analyzed by the practitioner or their staff, or it mined by knowing the sequence of the transcript, the noncod may be sent to an outside or independent laboratory. The ing strand, or the encoded protein. The nucleic acid sequence medical practitioner may be cognizant of whether the test is at that position in one or both alleles can be determined by a providing information regarding the patients f AR genes or 40 number of ways known to those of skill in the art. Such ways alleles as distinguished from the encoded proteins, or the include, but are not limited to, chain terminating sequencing, medical practitioner may be aware only that the test indicates restriction digestion, allele-specific polymerase reaction, directly or indirectly that the genotype of the patient reflects single-stranded conformational polymorphism analysis, the Gly389/Gly389 phenotype (“homozygous Gly” genetic bit analysis, temperature gradient gel electrophoresis, sequence), the Arg389/Gly389 phenotype ("heterozygous' 45 or ligase chain reaction. sequence), or the Arg389/Arg389 phenotype (“homozygous Alternatively, the BAR protein sequence may be evalu Arg’ or "homozygous wild-type' sequence). ated. In certain embodiments, the amino acid at position 389 Similarly, the medical practitioner may be cognizant of in one or more of the patient's BAR protein is known. It is whether the test is providing information regarding the contemplated that any sample evaluated from the patient will patient’s CAR genes or alleles as distinguished from the 50 contain multiple BAR proteins that can be analyzed. An encoded proteins, or the medical practitioner may be aware analysis of these proteins can determine if the patient has only that the test indicates directly or indirectly that the geno BAR proteins with only an arginine at 389, only a glycine at type of the patient reflects the homozygous wildtype 389, or a mixture of both types. Similarly, the CAR protein sequence (no deletion in either allele), the heterozygous sequence may be evaluated. In particular embodiments, Del322-325 phenotype ("heterozygous' sequence), or the 55 whether there is a deletion of amino acids 322-325 in one or Del322-325/Del322-325 phenotype (“homozygous deletion” more of the patient’s CAR protein is known. sequence). It is contemplated that any sample evaluated from the In Some embodiments discussed in the Examples, a patient patient will contain multiple BAR and CAR proteins that with either the heterozygous sequence or the homozygous may be analyzed. An analysis of these proteins can determine Gly sequence with respect to BAR is referred to as a “Gly 60 if the patient has BAR proteins with only an arginine at 389, carrier. Likewise, a patient with either the heterozygous only a glycine at 389, or a mixture of both types. Likewise, it sequence or the homozygous deletion sequence with respect may be determined whether the patient has CAR proteins to CAR is referred to a “Del322-325 carrier” or a “deletion with only the wildtype sequence at amino acids 322-325 (no carrier.” deletion), only a deletion of the amino acids corresponding to In any of these circumstances, the medical practitioner 65 322-325, or a mixture of both types. “knows the relevant information that will allow him or her to Methods for determining the sequence at a particular posi determine whether bucindolol is an appropriate medicinal tion in a protein are well known to those of skill in the art. US 8,080,578 B2 10 They may involve using an antibody, high pressure liquid prescribed bucindolol may have class III or class IV heart chromatography, or mass spectroscopy. failure according to the NYHA classification system. The As discussed above, the sequence of a particular position in NYHA classification system is one evaluation system, how the BAR gene or protein and/or CAR gene or protein may ever, it is contemplated that the invention is not limited in this be known. Some methods of the invention involve determin 5 way and that this is meant to be illustrative rather than limit ing the sequence in the BAR gene or protein sequence and/or ing. Patients may be classified by another Such system. It is CAR gene or protein sequence. further contemplated that patients may be classified by a Consequently, it is contemplated that embodiments may different methodology but that the invention would be imple involve obtaining a biological sample from a patient. A bio mented similarly. logical sample is a sample that contains biological material 10 In other embodiments, however, a patient may have signs Such as all or part of an organ, tissue, cells, nucleic acids, or symptoms of heart failure but not advanced heart failure. In proteins, or other such macromolecules and Substances. The Such a situation the patient may have been or may be charac sample may include sputum, serum, blood, plasma, spinal terized as a class I or II heart failure patient according to the fluid, semen, lymphatic fluid, urine, stool, pleural effusion, NYHA classification system. In these embodiments, the ascites, a tissue sample, tissue biopsy, cell Swab, or a combi 15 patient may be genotyped for the Arg389 B polymorphism, nation thereof. In other embodiments of the invention, a in which case a person with the Arg/Arg phenotype is a sample may include cells that are from lung, skin, muscle, candidate for bucindolol treatment. Consequently, methods liver, renal, colon, prostate, breast, brain, bladder, Small intes of the invention can involve preventing heart failure in a tine, large intestine, cervix, stomach, pancreas, testes, ova patient by determining whether the patient has Arg389/ ries, bone, marrow, or spine. In some embodiments, the Arg389 polymorphism and administering bucindolol if they sample is a whole blood, plasma or serum sample, while in do. Particular patients might be particularly suited for this other embodiments, the sample is obtained by lavage, Smear, including, but not limited to, those patients with symptoms of or Swab of an area on or in the patient. In certain embodi heart failure, with risk factors of heart failure, or with a ments, the biological sample is a blood sample. familial or prior history of heart failure. In some embodiments of the invention, the sequence of a 25 Additionally, methods may involve administering or pre patient's BAR genes and/or proteins and/or the sequence of scribing other therapeutic agents or performing a Surgical or a patient's CAR genes and/or proteins may already have other interventional strategy for treating the patient. been evaluated. It is contemplated that this analysis may have According to the present invention, methods may further been done prior to the patient being considered for treatment involve prescribing or administering bucindolol to the patient with bucindolol or as part of a general examination. For 30 after knowing that the patient’s genotype at the 389 polymor example, the sequence of the patient's BAR genes and/or phism is Arg389/Arg389, also known as the homozygous proteins, as well as his CAR genes and/or proteins may be arginine genotype. Additionally or alternatively, bucindolol determined and entered into a database or entered into the may be prescribed or administered after knowing that the patient's medical history. In this case, a medical practitioner patient has the homozygous wildtype CAR polymorphism may come to know what the sequence is by obtainingapatient 35 (does not carry a deletion of nucleotides 964-975 in either history regarding the sequence i) at position 1165 in the allele of the CAR gene). Moreover, it may be the case that a coding sequence of one or both BAR alleles orii) at position patient who does not exhibit either or both genotypes will not 389 in the amino acids sequence of the BAR protein; iii) at be prescribed or administered bucindolol. The patient may be position 964-975 in the coding sequence of one or both prescribed or administered a n-blocker that is specifically not CAR alleles; and/or iv) at positions 322-325 in the amino 40 bucindolol. acids sequence of the CAR protein. In additional embodiments, methods can involve knowing The present invention also involves reporting the results of whether there is a deletion in (iii) the nucleotide sequence at a determination of the nucleic acid or protein sequence at the positions 964-975 in the coding sequence of one or both of the relevant position in the BAR alleles or protein and/or the patient's CAR genes or (iv) the amino acid sequence at CAR alleles or protein. In certain embodiments, methods 45 positions 322-325 of one or more of the patient’s CAR include preparing a report containing the results of determin proteins. This may be known in addition to or independently ing (i), (ii), (iii), and/or (iv) described in the previous para of whether the patient’s genotype in the faR gene at posi graph. Such a report would identify the patient by name, tion 1165 (389 in the protein). Social security number, and/or other identification number or If an advanced heart failure (that is, NYHA class III or IV) qualifier. It may also contain the actual data as a result of the 50 patient exhibits a homozygous CAR Del322-325 genotype determination or a Summary of that data. and the patient does not exhibit the Arg389/Arg389 genotype, In some embodiments, methods include identifying a it is contemplated the patient will not be prescribed or admin patient possibly in need of treatment with a bucindolol. A istered bucindolol. In certain embodiments, the patient is patient for which bucindolol is being considered as a treat identified as a patient whose race is Black. Alternatively, if a ment option may have symptoms of or may have been diag 55 patient does not exhibit a homozygous CAR Del322-325 nosed with a medical condition, Such as heart failure, dilated genotype, the patient may be prescribed or administered cardiomyopathy, ischemic heart disease, pheochromocy bucindolol. toma, migraines, cardiac arrhythmias, hypertension or an Other information may also be considered in determining anxiety disorder. In certain embodiments, the patient has whether bucindolol is an appropriate drug for the patient. This symptoms of or has been diagnosed with ischemic heart dis 60 may include race, gender, age, previous Surgeries, heart fail ease, which may specifically be angina and/or a myocardial ure stage, patient history regarding cardiovascular disease, infarction. In particular cases, a patient has symptoms of or diagnosis of other diseases or conditions, risks for other dis has been diagnosed with heart failure. The heart failure may eases or condition, drug allergies, drug toxicity, and/or other be considered advanced heart failure, though the invention medications being taken. may not be limited to such patients. The term “advanced heart 65 Therefore, it is contemplated that the invention also con failure' is used according to its ordinary and plain meaning in cerns doing a diplotype analysis or obtaining the results of a the field of cardiology. In some embodiments, a patient being diplotype analysis. In particular embodiments, the diplotype US 8,080,578 B2 11 12 analysis involves evaluating directly or indirectly the poly FIG. 5. This illustrates the hazard ratios and 95% confi morphism (1) at position 389 of BAR so as to determine dence intervals for heart failure outcomes stratified by BAR whether a patient has an Arg389/Arg389 genotype and (2) at genotype. position 322-325 of CAR so as to determine whether the FIG. 6. This graph illustrates the survival of patients in the patient has a Del322-325/Del322-325 genotype. Other poly bucindolol-placebo study stratified by treatment and BAR morphisms may be included in the haplotype, particularly genotype. those that are affected or affect the patient’s ability to respond FIG.7. This graph illustrates the probability of reaching the favorably to bucindolol as a therapeutic agent. combined endpoint of death or heart failure hospitalizations Any embodiment discussed with respect to one aspect of in the bucindolol-placebo study stratified by treatment and the invention applies to other aspects of the invention as well. 10 BAR genotype. This includes embodiments discussed with respect to each of FIG.8. Change from baseline norepinephrine levels: SEM BAR and CAR. Specifically, any embodiment discussed at 3 months and 12 months, by treatment group. In both A and with respect to BAR genes, alleles, or protein may be imple BThe numbers under the bars are the numbers of patients in mented with respect to CAR genes, alleles, or proteins, and 15 each group who had baseline and interval measurements at Vice versa. each timepoint; p values are for a comparison of change in The embodiments in the Example section are understood to each treatment group. be embodiments of the invention that are applicable to all FIG. 9. Hazard ratios relative to the first quartile for all aspects of the invention. cause mortality, by quartile of baseline norepinephrine. The The use of the term 'or' in the claims is used to mean norepinephrine cut points defining quartiles are: 1, s304 “and/or unless explicitly indicated to refer to alternatives pg/ml. 2", 305 pg/ml to 436 pg/ml: 3', 437 pg/ml to 635 only or the alternatives are mutually exclusive, although the pg/ml. 4", 2636 pg/ml. Numbers of patients per quartile are: disclosure Supports a definition that refers to only alternatives Placebo group 1' 294, 2' 255, 3 248, 4', 264; Bucindolol and “and/or.” group 1' 239, 2'274, 3284,4', 267. Throughout this application, the term “about is used to 25 FIG. 10. Hazard ratios relative to the first quartile for the indicate that a value includes the standard deviation of error combined endpoint of all-cause mortality+CHF hospitaliza for the device or method being employed to determine the tion, by quartile of baseline norepinephrine. value. FIG. 11. Likelihood analysis for change in norepinephrine Following long-standing patent law, the words 'a' and at 3 months vs. Subsequent all-cause mortality, Placebo and “an.” when used in conjunction with the word “comprising 30 Bucindolol treatment groups. FIG. 12. Nonfailing and failing human ventricular ex vivo in the claims or specification, denotes one or more, unless contractile responses correlate with BAR genotype. Right specifically noted. ventricular trabeculae were utilized from nonfailing and fail Other objects, features and advantages of the present ing human hearts as described in Methods. Trabeculae from invention will become apparent from the following detailed 35 11 hearts were studied in each of the four groups. All Arg strps description. It should be understood, however, that the were from homozygous Subjects. The Gly carriers consisted detailed description and the specific examples, while indicat of 10 hetoZygotes in the nonfailing and 9 in the failing groups, ing specific embodiments of the invention, are given by way with the remainder being homozygotes for Gly. The maximal of illustration only, since various changes and modifications response derived from the dose response curves was greater within the spirit and scope of the invention will become 40 for Arg389, both in the nonfailing (P=0.01) and failing apparent to those skilled in the art from this detailed descrip (P=0.008) studies. tion. FIGS. 13 A-D. Effect of increasing doses of bucindolol or Xamoterol on peak systolic force (mN/mm) in isolated right BRIEF DESCRIPTION OF THE DRAWINGS Ventricular trabeculae from failing human hearts. Dose-re 45 sponse curves were performed without (bucindolol, panel A. The following drawings form part of the present specifica Xamoterol, panel C) and with (bucindolol, panel B: Xamot tion and are included to further demonstrate certain aspects of erol, panel D) pretreatment by 10 M forskolin to enhance the present invention. The invention may be better understood B-AR signal transduction. In the forskolin pretreatment by reference to one or more of these drawings in combination experiments, forskolin-alone trabeculae were allowed to with the detailed description of specific embodiments pre 50 incubate throughout the treatment period, and any effects on sented herein. force were subtracted from the bucindolol or Xamoterol FIG. 1. The chemical structures of several B-blockers, treated trabeculae. *, p<0.05 vs. baseline tension; it, p<0.10 including bucindolol, is depicted. vs. baseline tension; S, p<0.05 vs. slope of 0 for entire curve: FIG. 2. Comparison chart of different anti-adrenergic I, p<0.05 vs. slope of 0 for doses of 10M to 10 M:p values agents and treatments based on Phase II or III heart failure 55 associated with brackets are for test for interaction between clinical trial data or other development data. curve slopes, with 1st p valur for doses between 10 M to FIG. 3. This figure illustrates the allele-specific effects of 10M, and 2nd p value for entire curve. bucindolol in cells stably expressing BArg389- or BGly389. FIG. 14. Antithetical consequences of chronic myocardial Results are meant SE of 4 experiments. B adrenergic stimulation. FIG. 4. Bar graph illustrates a response to B-blockade in 60 FIG. 15. Characteristics of norepinephrine change in risk transgenic mice with targeted overexpression of Gly389 and groups and patients treated with bucindolol. Standard devia Arg389 BAR to the heart. Shown are mean (SE) results tions (SD) are included in chart. from Western blots for the indicated proteins from hearts of FIG. 16. Additional characteristics of norepinephrine B-Arg389 and B-Gly389 mice (n=3-4 in each group). Data change in risk groups and patients treated with bucindolol. are normalized to the control (untreated) values. An overall 65 Standard deviations (SD) are included in chart. treatment response to propranolol was found only in hearts FIG. 17. Illustration of correlation between CAR geno from the B-Arg389 mice (P<0.002 by ANOVA). type and systemic norepinephrine levels in BEST patients. US 8,080,578 B2 13 14 FIG. 18. Illustration of correlation between CAR geno is denoted BEST (B-blocker Evalutation Survival Trial). In type and systemic norepinephrine response (pgtSEM) in the transfected cells, functional antagonism by bucindolol of BEST patients after three months. norepinephrine-stimulated cAMP was assessed. Even though FIG. 19. Correlations between CAR genotype, systemic the Arg389 receptor displayed markedly higher norepineph norepinephrine response (pg|SEM) in BEST patients after 5 rine-stimulated cAMP production, bucindolol antagonized three months, and survival by treatment group among BEST the response. The absolute decrease in cAMP production was patients. substantially greater for Arg389 vs Gly389 expressing cells, FIGS. 20A-D. Effect of BAR Arg/Gly 389 gene variants which is due to the high norepinephrine stimulation of on isoproterenol response in isolated human RV trabeculae. Arg389 and the efficacy of bucindolol to fully antagonize the A. Homozygous BAR Arg 389 failing Iso response. B. 10 response. Approximately 80% inhibition of the Arg389 B-AR Gly 389 carrier failing Iso response. C. Homozygous cAMP response was observed at 0.1 uMbucindolol, which is f3AR Arg 389 nonfailing Iso response. D. BAR 389 Gly comparable to plasma concentrations of the drug at the doses carrier nonfailing Iso response. used in BEST (unpublished data). These results suggested FIG. 21. CAR and BAR gene variants: treatment effects that in patients a greater change in cardiac faRactivity from in BEST (mortality). 15 bucindolol treatment might be possible in those with the FIG. 22. B-blocker mortality trials in U.S. populations, f3-Arg389 compared to the B-Gly389 genotype, and poten mortality hazard ratios 95% C.I.s. tially result in a more favorable clinical response. In the FIG. 23. Effect of B-blocking agents on systolic function in transgenic mouse studies, the inventors examined the effect NF hRV trabeculae. of B-blockade over a 6 month period on the expression of key FIG. 24. Effect of B-blocking agents on systolic function in signaling and Ca"-handling proteins in the heart. With the NF hRV trabeculae with amplification of signal transduction Gly389 mice, there was no effect of treatment on expression by 10 uM forskolin (F) treatment. of these proteins. On the other hand, an overall treatment FIG. 25. Effect of B-blocking agents on systolic function in effect from B-blockade was noted in the Arg389 mice, with failing hRV trabeculae. changes that are consistent with reverse remodeling at the FIG. 26. Effect of B-blocking agents on systolic function in 25 molecular level. Next, the archived DNA from BEST, a study failing hRV trabeculae with amplification of signal transduc which provided extensive phenotyping and matched placebo tion by 10 LM forskolin (F) treatment. group, was utilized; due to the transfected cells and transgenic FIG. 27. Study Design I. mice results, the inventors had an a priori hypothesis that FIG. 28. Study Design II. B-Arg389 would be the most favorable genotype for sur FIG. 29. Study Design III. 30 vival. Here, a dominant model was assumed. Thus, the two genotype groups were Arg389 homozygotes and patients DESCRIPTION OF ILLUSTRATIVE with one or two Gly389 alleles (i.e., homozygous for Gly or EMBODIMENTS heterozygotes; this group is termed “Gly389 carriers'). The clinical endpoint results from BEST indicate no mortality, The inventors of the present invention were confronted 35 heart failure hospitalization or mortality+heart failure hospi with the data that bucindolol appeared to provide less favor talization benefit of bucindolol treatment in patients who are able therapeutic responses in certain patient subgroups and a B-Gly389 carriers, but clinically relevant improvements in less favorable response than other f-blockers by certain cri all three outcomes in B-Arg389 homozygous patients treated teria. They hypothesized that the interindividual variability in with bucindolol as compared to placebo. Baseline clinical the response to bucindolol in heart failure (HF) is due to 40 parameters including heart rate, blood pressure and LVEF, or genetic variability and determined the basis for this. In doing the etiology of heart failure, were not predictive of endpoint So, the inventors were able to make a significant case for the response in the entire cohort that included all faR gene therapeutic value ofbucindolol and its appropriateness for the variants. Furthermore, there was no apparent effect of the treatment of heart failure in humans. B-Gly49 polymorphism on these relationships. Taken The genetic variability of the BARatamino acid 389 of the 45 together, then, the results from these studies strongly suggest protein (nucleic acid 1165 of the gene) was evaluated. This that position 389 variant of BAR is a predictor of the was based on the properties of the two receptors, denoted here response to bucindolol in heart failure. as Arg389 and Gly389, as ascertained in transfected cells, Moreover, a role for the genetic variant in the C.2cAR gene where basal and agonist-stimulated adenylyl cyclase activi was also postulated for bucindolol efficacy. The inventors of ties are approximately 3-fold greater for the Arg receptor 50 the present invention hypothesized that the interindividual (Mason et al., 1999). However, prior to the current studies, it variability in the response to bucindolol in heart failure is due was not clear whether patients with Arg389, or Gly389, to genetic variability of the CAR gene. The present inven would benefit most from bucindolol treatment. For example, tion thus approached the question of whether the ODel322 the enhanced function of Arg389 may have made it impos 325AR allele represents a pharmacogenetic locus for predict sible for bucindolol to act as an effective antagonist. The 55 ingresponse to bucindolol in heart failure using BEST, a large ultimate test of the hypothesis, where death, cardiac trans multicenter placebo-controlled trial (see Examples discussed plantation or HF hospitalizations (heart failure exacerbations in detail below). The archived DNA from BEST, a study that in actual patient treated with placebo or bucindolol) were provided extensive phenotyping and matched placebo group, evaluated, had not been carried-out. was utilized. Here, a dominant model was assumed. Thus, the The present invention thus approached the question of 60 two genotype groups were 1) C wild-type homozygotes whether the Arg (or Gly) 389 (BAR allele represents a phar (patients with no deletion in 322-325 on either allele) and macogenetic locus for predicting response to (B-blockers in ODel322-325 heterozygotes or homozygotes (patients with heart failure using a three-tiered approach involving investi the deletion in one or both alleles, referred to as “ODel322 gations in transfected cells (see Example 1 discussed in detail 325 carriers'). The clinical endpoint results from BEST indi below), transgenic mice (see Example 2 discussed in detail 65 cate no mortality, heart failure hospitalization or mortality below) and a large multicenter placebo-controlled clincal trial plus heart failure hospitalization benefit of bucindolol treat (see Example 3 discussed in detail below). The clinical study ment in patients who are C. Del322-325 carriers, but clini US 8,080,578 B2 15 16 cally relevant improvements in all three outcomes in C. transfected to express equal levels of the two receptors, the wild-type homozygous patients treated with bucindolol as f-Arg389 receptor display substantially greater stimulation compared to placebo. Baseline clinical parameters including of adenylyl cyclase compared to B-Gly389 (Mason et al., heart rate, blood pressure and LVEF, or the etiology of heart 1999). A less common polymorphism of the BAR, Gly49, failure, were not predictive of endpoint response in the entire has also been identified but there are discrepant reports as to cohort that included all CAR gene variants. Taken together, its functional implications (RathZ. et al., 2002; and Levin et then, the results from these studies strongly suggest that the al., 2002). ODel322-325 polymorphism is a predictor of the response The B-AR389 Arg/Arg polymorphism is actually the most to bucindolol in heart failure. prevalent form of the fadrenergic receptor and is present in Therefore, the present invention concerns methods that 10 about 50% of the U.S. population (slightly less in African utilize the genetic relationship between the Arg389 BAR Americans). The other variant of this receptor has a glycine polymorphism and bucindolol therapy and between the (Gly) at the 389 position and is considered the wild type only Del322-325 CAR polymorphism and bucindolol therapy. because it was cloned first. The presence of an arginine (Arg) I. Adrenergic Receptors and B-Blockers at codon 389 is the preferred (and only) structure of this Treatment for heart failure has involved targeting adrener 15 receptor in all other known non-human animal species, and gic receptors (AR). There are at least nine Sub-types of adr the 389 region is in an important functional domain. The energic receptors (Dohlman et al., 1991; and Liggett et al., 389 Arg/Arg is also the highest functioning variation of this 1993), of which at least three sub-types are f3-adrenergic receptor (Mason et al., 1999); its signal transducing efficacy receptors. is 3-4 times greater than for Gly heterozygotes or Gly/Gly at A potential role for common genetic variants in Suscepti the 389 position (see Examples and Mason et al., 1999). The bility, progression and response to treatment is suggested by increased signal transduction capacity of the f-AR389Arg/ familial clustering of phenotypes, reduced penetrance in Arg applies to cAMP generation (Mason et al. 1999), isolated familial cardiomyopathies and marked interindividual varia human heart muscle contraction (Mason et al. 1999), and tions in progression and treatment outcomes. While polymor production of cardiomyopathy in transgenic mice (Mialet phisms in adrenergic receptors have been identified, there has 25 Perez et al., 2003). been no study involving patients data in which a correlation Certain B-blockers have been evaluated in the context of between any polymorphism and a clinical response to a thera specific genetic variations with varying results. Sofowora et peutic agent has been identified. The present invention con al. reported that patients who are homozygous for Gly389 are cerns two polymorphisms: 1) the polymorphism encoding the less sensitive to the effects of atenolol, a selective B-adrener amino acid at position 389 in B-AR and 2) the polymorphism 30 gic receptor, based on hemodynamic responses, Suggesting to encoding amino acids 322-325 in C-AR. However, the rela the authors that the variation may be relevant particularly to tionship between these particular genetic variant and any resting blood pressure responses. Johnson et al. (1993) treatment outcome had not been established with any clinical reported that homozygotes for Arg389 were more likely to evidence prior to the present invention, nor had any correla respond to metoprolol, a selective (B-blocker, as measured by tion been demonstrated with bucindolol. 35 blood pressure. Perez et al. (2003) evaluated position 389 A. B. Adrenergic Receptor variants of the BAR in the context of intact cardiac function The B adrenergic receptor (B-AR) is the principle Sub using targeted transgenesis in mice. In these transgenic mice type expressed on cardiac myocytes. The human heart overexpressing either homyzgous Arg or Gly at the 389 posi expresses both the AR and the BAR subtypes (Bristow et tion, Arg/Arg mice had a greater loss ofisoproterenol respon al, 1986; Bristow et al., 1988). Each receptor mediates posi 40 siveness for increases in myocardial function, and a greater tive inotropic and chronotropic responses to endogenous cat degree of cardiomyopathy as measured by myocardial dys echolamines and exogenously administered agonists (Bris function, degree of chamber remodeling, and histology. They tow et al., 1986; Brodde et al., 1986; Brodde et al., 1992). also reported a greater improvement in left ventricular func The BAR triggers the heart’s contractile response when tion in patients treated with carvedilol, a non-selective activated, as it is by norepinephrine. In addition, the B recep 45 B-blocker, that was associated with the Arg389 polymor tor has a central role in the progression of cardiomyopathy phism, in either the homozygous or heterozygous state. and other disease pathways. Increased activation of this Liu et al. (2003) report finding that a greater response (in receptor and its associated myopathic and arrhythmic path terms of changes in heart rate) to metoprolol was associated ways plays a major role in the natural history of heart failure. with Arg389 compared to Gly389. The authors also warned Once the cardiomyopathic process has begun, chronic B-adr 50 about extending the results beyond their patient pool, which energic activation accelerates disease progression, as the fail was healthy, young, male Chinese Volunteers. They specifi ing heart tries to compensate for its impaired functioning by cally say that the study did not look at any long-term effects of releasing more norepinephrine and increasing 3-receptor metoprolol with respect to the polymorphisms. signaling. The theory of B-receptor blockade rests in part on A review article published in 2004.(Lohse, 2004) noted that counteracting this cardiomyopathic pathway by blocking the 55 while the Arg389 polymorphism might be relevant to the f-receptor and reducing norepinephrine signaling. benefit from treatment of B-blockers, there had been no study The Badrenergic receptor has been cloned and sequenced regarding the influence of 3-adrenergic receptor polymor (Frielle et al., 1987). The gene has been localized to chromo phisms on responsive to B-blockers in heart failure. Another some q24-q26 of chromosome 10 (Yang-Feng et al., 1990). review indicated that while some studies suggested that poly The human fBAR has a deduced amino acid sequence of 477 60 morphisms in adrenergic receptors might alter the response to amino acids. treatment with B-blockers, firm conclusions or recommenda At coding nucleotide position 1165 of the BAR gene, tions for patient management could not be made because of either cytosine or guanine can be found in the human popu the low patient numbers in the different studies lation, which results in either Arg or Gly being encoded at Moreover, several reports did not detect any correlation amino acid position 389 (Mason et al., 1999). This position is 65 between a polymorphism at 389 of the f-AR and the treat within an intracellular domain of the receptor that is involved ment response to a B-blocker. Thus, there is a distinguishing with coupling to the stimulatory G-protein, G. In fibroblasts point with respect to the present disclosure, although the US 8,080,578 B2 17 18 specification points to a reference of White et al. (see argu polymorphism is much more common in African-Americans, ment #1 below), which allegedly looked at metoprolol with a 0.4 allele frequency compared to 0.04 in non-African treated heart failure patients as assessed by mortality or the Americans. Overall, this polymorphism is present in about combined endpoint of mortality+heart failure hospitalization, 15% of the U.S. population. and found no association. O'Shaughnessy et al. (2000) In contrast to the Arg389 polymorphism, the 322-325 Del reported that no difference was seen in blood pressure or heart polymorphism of the Cadrenergic receptor is an uncom rate response to beta blockade (atenolol or bisoprolol both mon and low-functioning variation of this receptor (Small et antagonists) due to 389 polymorphism. Another paper, al., 2000). Loss of these four residues predicts a reduction in Joseph et al. (2004), also observed no difference in receptor receptor function, which is supported by cellular transfection affinity for B-blockers with 389 polymorphism. Furthermore, 10 experiments where receptor function is curtailed by 50-85%. a recent study reported being unable to find any evidence of “a (Small et al., 2000). The O receptor ordinarily tonically pharmacogenetic effect” on metoprolol treatment with inhibits norepinephrine release prejunctionally in adrenergic respect to the Arg389 polymorphism. White et al., Eur: J. nerve terminals (Hein et al., 1999). Heart Fail. 5:463-8, 2003. The 322-325 deletion essentially destroys receptor func Therefore, a correlation between the effectiveness of 15 tion (Small et al., 2000) leading to higher levels of norepi B-blockers as a class of therapeutic agents and the 389 poly nephrine and adrenergic drive (Neumister et al., 2005). The morphism in B-AR had not been established. Furthermore, consequence of diminished inhibitory control is that basal no correlation could be made regarding bucindolol particu norepinephrine release is constitutively increased resulting in larly, which differs from the other B-blockers in several a higher state of sympathetic activity. (Hein et al., 1999). In important respects. Because the differences among the vari heart failure this becomes of particular interest as O'Del322 ous B-blockers is significant, the effects of the BAR-389 325 receptors lack the “protective' braking effect against variants on 3-blocker response may be dependent on the increased sympathetic drive. specific agent. The ODel322-325 receptor polymorphism, present in The present disclosure provides data that when the BEST one study as a homozygous genotype in only 7.4% of Cau data is evaluated in the context of individual genotype, par 25 casians but in 52.6% of Blacks with chronic heart failure, ticularly at the Arg389 polymorphism, bucindolol has sub results in loss of function as assessed by inhibition of adenylyl stantial therapeutic efficacy. This data is Surprising given that cyclase stimulation (Small et al., 2002). This defect has func in the BEST study the mortality effects observed in the total tional consequences on C-AR function and has been inter population studied were lower than what had been observed changeably referred to as both “polymorphism' and a “muta with other n-blockers such as carvedilol, metoprolol CR/XL 30 tion” reflecting the characteristics of relative commonness in and bisoprolol. Furthermore, the scientific data provided populations and its profound structural/functional effects, herein demonstrate for the first time a correlation between the respectively. Throughout this proposal the variant is referred therapeutic efficacy ofbucindolol and two genetic variaants. to primarily as a “polymorphism' to reflect that this is a In detail, the invention provides a method for determining common variant present in many Subjects with heart failure. whether bucindolol should be prescribed to a patient wherein; 35 Its importance, however, stems from its functional conse the identity of a polymorphic nucleotide oramino acid site of quences, profoundly diminishing receptor activity. Based on a BAR and a C-AR is determined and based on the results the effects of genetic ablation in mice, the wild type, fully of that diagnostic test bucindolol is either prescribed or not. functional C receptor is prejunctionally inhibitory to nore Similarly, based on the genotype, another medication may be pinephrine release; as the knockout mice display a loss of this prescribed for patient with the unfavorable BAR genotype, 40 inhibition that leads to increased norepinephrine levels and So as to attempt to gain improved clinical response. In both pathological hypertrophy. (Hein et al., 1999). The clinical scenarios, drug treatment decisions are based on the faR importance in humans of this polymorphism was illustrated a genotype of the patient. study that identified the ODel322-325 genotype as a risk Thus, the invention concerns methods for evaluating bucin factor for heart failure; having an unadjusted odds ratio for dolol therapy for a patient, particularly a heart failure patient, 45 heart failure of 5.54 (95% CI 2.68, 11.45; p<0.001) in Blacks based on whether the individual is homozygous Arg389 at the homozygous for this genotype as compared to heterozygotes fAR gene, homozygous for the wildtype form of the CAR and noncarriers. (Small et al., 2002). In conjunction with the at amino acid position 322-325, or both. Alternatively, the BArg389 variant the effect was more pronounced with an present invention concerns a method concerning the diplo unadjusted odds ratio of 12.67 (95% CI 2.70, 59.42: type of BAR389Gly carrier and CARDel322-325 carrier. 50 p=0.001). However, in contrast to the present disclosure, B. C. Adrenergic Receptor there has been no indication that these polymorphism are The C-ARS are located on cardiac sympathetic nerve relevant from a therapeutic perspective. terminals, and regulate the prejunctional neuronal release of C. B-Blockers norepinephrine into the synaptic cleft area. Binding of nore While Bagonists such as dobutamine, are used for treating pinephrine to C-ARS invokes a negative feedback sym 55 acute deterioration of patients with failing ventricular func patholytic response that attenuates further neuronal norepi tion, prolonged exposure of the heart from administered ago nephrine release. Murine gene ablation models implicate the nists, or the elevated endogenous catecholamine agonists pro C-ARS as being principally responsible for controlling the duced by the body, leads to worsening heart failure. Indeed chronic steady rate of norepinephrine release. (Hein et al., BAR and BAR become desensitized in heart failure, which 1999). In this way the C-AR has a “protective' role in the 60 is thought to be a mechanism of self-protection against the heart, buffering against the chronically elevated levels of high levels of catecholamines that exist in heart failure. The norepinephrine encountered in the failing human heart. administration off antagonists can improve ventricular func A human genetic polymorphism has been reported for the tion and clinical outcomes, presumably by blocking these C.2c-AR gene, ADRA2C. (Small et al., 2000). A loss of 12 deleterious effects of catecholamines. And indeed, cardiac nucleotides in ADRA2C translates to a deletion of four con 65 BAR expression and function improve during B blockade secutive amino acids (ODel322-325) in the third intracellu treatment of heart failure. The vast majority of the deleterious lar loop of the receptor. (Small et al., 2000). This deletion effects of catecholamines, and the success of B blocker US 8,080,578 B2 19 20 therapy is due to variants of the BAR subtype. (Zhu et al., or III clinical trials depicts these differences. Carvedilol, for 2001; and Bristow et al., 2003). instance, is an efficient f-AR and f-AR blocker, as well as B-adrenergic receptor antagonists (also termed (B-block an C-AR blocker. In contrast, bucindolol is a weak C-AR ers) have emerged as a major treatment modality in chronic blocker, and metoprolol and bisoprolol do not block C.-AR at heart failure. Initially these agents were thought to be con all. Significantly, bucindolol is unique among B-blockers in traindicated in heart failure, since increased adrenergic drive its sympatholytic properties, in contrast to carvedilol, meto was thought to be critical for Supporting the failing heart. In prolol, and bisoprolol, which have no such properties. Com fact, in early experience with the 1 generation compound pared to other 3-blocking agents bucindolol uniquely lowers propranolol, administration of standard doses was frequently systemic norepinephrine levels (Lowes et al., 2000; Bristow associated with worsening of heart failure (Stephen, 1968). 10 et al., 1997: BESTNEJM, Bristow, 2004), and is a full agonist However, using low starting doses and slow up-titration, 2" for the B-adrenergic receptor (Strosberg, 1997). generation (selective f-blockers) or 3" generation (nonse Bucindolol is a 3rd generation, B-blocker-vasodilator with lective B-blocker-vasodilators) generation compounds have the chemical name and structure of (2-2-hydroxy-3{{2-(3- been shown to reverse contractile dysfunction as well as indolyl)-1,1-dimethylethylaminopropoxy-benzonitrile structural and molecular remodeling, and to improve heart 15 hydrochloride). It was first developed for hypertension, and failure morbidity and mortality (Bristow, 2000): CIBIS-II then for heart failure. Because of its low inverse agonist and Investigators and Committees. The cardiac insufficiency vasodilator properties the nonselective B-blockade of bucin bisoprolol study II: a (CIBIS-II, 1999); MERIT-HF Study dolol is relatively well tolerated by heart failure patients, and Group. Effect of metoprolol CR/XL in chronic heart failure: in part for this reason in 1994 bucindolol was selected by the Metoprolol CR/XL Randomized Intervention Trial in Con NIH and VA Cooperative Clinical Trials Group to test the gestive Heart Failure (MERIT-HF, 1999). Packer et al. hypothesis that a B-blocker could reduce mortality in (2001); BEST Trial Investigators, (2001); Lowes et al., 2002). advanced heart failure. The test of this hypothesis was the In part, these beneficial effects are thought to be due to a BEST Trial, which was conducted between May 31, 1995 and protection of the failing heart, which has limited metabolic Jul. 29, 1999. and physiologic reserves, from persistent adverse biological 25 The Beta-blocker Evaluation of Survival Trial (“BEST) effects mediated by elevated norepinephrine levels found in was stopped prematurely on recommendation of the Data and the syndrome (Bristow, 2000; Cohn et al., 1984; and Liggett, Safety Monitoring Committee, at a time when the hazard ratio 2001). In addition, B-blockers have been shown to partially for the primary endpoint of all-cause mortality was 0.90 (C.I.s reverse the molecular phenotype of heart failure (Lowes et al., 0.78-1.02) (BEST Investigators, 2001; Domanski et al., 2002), so these agents are capable of both preventing and 30 2003). However, the results for the entire BEST cohort were reversing progressive myocardial failure and remodeling positive for the high order secondary endpoint of mortality or Eichhorn and Bristow, Circulation 1996). heart failure hospitalization, which was reduced by bucin Interestingly, recent studies have shown that the heart rate dolol by 19% with a p value of <0.0001 (Domanski et al., and/or blood pressure response to the B-blockers metoprolol 2003). This endpoint is in fact increasingly viewed as the and atenolol is greater in normotensive Arg389 individuals 35 preferred primary endpoint for HF pivotal trials. compared to Gly389 individuals (Liu et al., 2003; and Sofo The reasons why BEST was stopped were 1) confirmation wora et al., 2003). And, in hypertensives the blood pressure by BEST Trial data generated in Class III, non-Black patients response to metoprolol is greater in Arg389 compared to of the then recently published information from CIBIS-II Gly389 patients (Johnson et al., 2003). One published study (CIBIS Investigators, 1999) and MERIT-HF (MERIT-HF in heart failure has found no apparent association or trend 40 Study Group, 1999) trials that these types of heart failure between BAR polymorphisms and the combined response of patients have a substantial survival benefit from B-blockade, hospitalizations and death to metoprolol treatment (White et 2) increasing loss of equipoise among investigators, who al., 2003). In this study, though, metoprolol-treated patients believed that the efficacy of B-blockade in heart failure had were not directly compared to placebo patients by genotype, been demonstrated, and 3) inefficacy and trends toward and approximately 45% of the patients had mild heart failure 45 adverse events in subgroups (Class IV and Blacks) that had (NYHA Class II), and the mean follow-up period was only 12 not been previously investigated in n-blocker heart failure months. Such differences may account for this potential dis trials. Further development of bucindolol was then aban crepancy. However, bucindolol and metoprolol have some doned because it was not clear bucindolol could be success notable differences in their pharmacologic properties (Bris fully marketed, even if approved. tow, 2000; and Bristow et al., 1997). In particular, bucindolol 50 Therefore, in this large survival trial in which the endpoint lowers norepinephrine, dilates the peripheral vasculature, and evaluation was overall survival, the BEST clinical trial was more potently blocks the human B-adrenergic receptor. terminated early because of confirmation of benefit that had While a common pharmacologic property of all B-blocking recently been shown in other trials, and the inability to extend agents that have been used to treat HF is that they block the the efficiacy of bucindolol to patient subgroups that had not BAR, which in the failing human heart has been estimated to 55 been previously evaluated in large scale clinical trials (BEST transduce up to approximately 90% of the pathologic adren Investigators, 2001). At that time, there was no significant ergic stimulation (Zhu et al., 2001; and Bristow et al., 2003), difference in mortality observed between those treated with the available f-blockers have a number of distinguishing bucindolol or with a placebo. In distinct contrast to the results properties including BAR-subtype selectivity, affinity for of BEST, similar studies with the B-adrenergic antagonists CAR, partial agonist activity, sympatholysis (Bristow et al., 60 bisoprolol (termed "CIBIS-II trial), metoprolol (termed 2004) and vasodilation (Bristow, 2000; and Bristow et al., “MERIT-HF trial), and carvedilol “COPERNICUS trial) 1997). reported very favorable differences (34-35% reductions in The chemical structure of some f-blockers is provided in mortality) between those treated with the antagonists and FIG. 1, which shows that these agents have significant struc those treated with a placebo. The BEST investigators specu tural differences. Moreover, they have different pharmaco 65 lated that one possible explanation for the difference in the logical properties. As is shown in FIG. 2, a comparison of results “may derive from the unique pharmacological prop different anti-adrenergic agents in development or in Phase II erties of bucindolol.” US 8,080,578 B2 21 22 In the CIBIS-II trial, the study was also stopped early, but However, when exaggerated, sympatholysis can be harmful, because the mortality rates were significantly less in those and can increase mortality (Bristow etal. 2004). As discussed treated with bisoprolol. CIBIS-II Investigators, 1999. Simi below, genetic targeting ofbucindolol allows this property to larly, in the MERITHF study with metoprolol, the study was function only in a favorable manner. ended prematurely because the predefined criterion had been 5 The second pharmacologic property of bucindolol that met and exceeded. MERIT-HF Study Group, 1999. The interacts with a pharmacogenetic target is high affinity B-re COPERNICUS study involving carvedilol was also halted ceptor blockade (Hershberger et al., 1990; Asano et al., 2001). early because of the significant benefits observed with treat Bucindolol has high affinity for human B-receptors, as well ment. Packer et al., 2001. The BEST investigators noted that as for B-receptors (Hershberger et al., 1990). In addition, their results raised questions about the equivalency of 10 through a non-agonist effect on either translation or protein B-blockers. Moreover, in previous non-mortality studies with turnover, bucindolollowers f-receptor density (Asano et al., carvedilol (Yancy et al., 2001), no response differences were 2001). Because it is so well tolerated, bucindolol can be observed between black and non-black subjects, which is administered at very high B-blocking doses, and each of these another specific, and relevant distinction with respect to 15 properties contributes to its salutary effects on the high func bucindolol. In the BEST trial, black patients with advanced tioning human B-receptor 389Arg/Arg gene variant (EX heart failure showed a worse outcome than non-blacks. Bris amples, Mason et al., 1999). Although bucindolol has intrin tow, 1997. sic sympathomimetic activity (USA) in rat myocardium in One review of the different trials stated, “No clear expla functioning human cardiac tissue bucindolol is devoid of ISA nation can be proposed for the reduced benefit obtained with (Bristow et al., 1994; Sederberg et al., 2000; Bristow et al., bucindolol in the BEST study.” Bouzamondo et al., 2001 1998, Example 7). This can clearly be seen in FIG. 13, panels (finding that if the BEST trial is excluded, the evidence indi A and B, where no significant increase in force development cates risk reduction achieved with B-blocker treatment). occurs in isolated failing human right ventricular trabeculae, While the authors say their study suggests that different heart even in the presence of signal transduction augmentation with failure populations subgroups have a different response to 25 the diterpene compound forskolin, in either the BAR Arg/ B-blocker therapy, they do not exclude the possibility that the Argor Gly carrier genotypes. In contrast, as shown in FIG. 13 different f8-blockers have different properties, nor do they say panel C, Xamoterol as a positive control ISA compound that polymorphisms are the reason. See also Sallach et al., exhibits an increase in force in both low and high signal 2003. (“While some authorities have suggested that the dif transduction activation in the BAR Arg/Arg genotype, but ference with the BEST trial was due to the patient population 30 only in the high activation state rendered by forskolin pre examined, others feel that the lack of mortality reduction is treatment in Gly carriers. Finally, as shown in FIG. 13, in due to bucindolol itself). preparations of isolated human heart, bucindolol has unique Therefore, there are therapeutic differences between effects on BAR Arg/Arg vs Gly carrier receptors. Under bucindolol and other B-blockers, and there was a significant conditions of low levels of signal transduction (low receptor question regarding the therapeutic efficacy of bucindolol 35 activation) in the failing heart (Panel A), bucindolol functions overall. Consequently, any relationship between bucindolol as a neutral antagonist (no agonist or inverse agonist activity) and particular genetic variants was not evident. at the human myocardial BArg/Arg receptor, but when signal The benefit of retrospective analysis based on the genetic transduction is high as when adenylyl cyclase is directly data disclosed herein highlights the unique pharmacologic activated by forskolin (Panel B), bucindolol functions as an features of bucindolol that contribute to its effectiveness in 40 inverse agonist, inactivating the receptor as indicated by a treating heart failure patients. Two of these features are also statistically significant slope factor up to the highest concen instrumental in the interaction of the drug with the adrenergic tration achievable in plasma by therapeutic doses, 10 M. No receptor gene variants. such effect occurs in Gly carrier receptors, where bucindolol The first of these features is sympatholysis, or the ability of functions as an inverse agonist in low activation states, and a a drug to lower adrenergic drive directly (lower norepineph 45 neutral antagonist in the presence of forskolin. These data rine levels in blood and tissue). As noted above, among Suggest that bucindolol is uniquely effective in antagonizing B-blockers that have been used to treat heart failure, bucin high activation states of the B 389Arg/Arg receptor, the form dolol is unique in this regard (BEST Trial Investigators, 2001; of the receptor that would be expected to be the most cardi Lowes et al., 2000; Bristow et al., 2004). The sympatholytic omyopathic. effects of bucindolol are likely due to B-receptor blockade 50 These properties are likely reasons for the Surprising and coupled with not enough C-blockade to activate adrenergic unexpected results that were observed with the Arg389 drive, and low inverse agonist activity for B- and B2-recep genetic variant in the BAR and the Del322-325 genetic vari tors to minimize adrenergic activation based on myocardial ant in CAR in the context of bucindolol treatment. depression. Other properties ofbucindolol that could contrib II. Analysis of Polymorphism ute to sympatholysis are nitric oxide generation and 3-re 55 Because the genetic variants are in coding regions of the ceptoragonism (Strosberg, 1997). These latter two properties f-AR and C.-AR genes and affect the encoded protein, the plus or minus a weak C-receptor blockade likely account for presence of the Arg,389 or Del322-325 polymorphism can be the mild vasodilator properties of bucindolol (Gilbert et al., determined from either the sequence of the nucleic acid or the 1990) which, unlike carvedilol, are not powerful enough to protein. As a result, a variety of different methodologies can trigger reflex adrenergic activation. 60 be employed for this purpose. When present in modest amounts, (Smaller reductions in A. Nucleic Acids norepinephrine) sympatholysis is a favorable property, con Certain embodiments of the present invention concern tributing to the therapeutic anti-adrenergic effect of bucin various nucleic acids, including amplification primers, oligo dolol. This is a potentially Superior mechanism of action to nucleotide probes, and other nucleic acid elements involved simple B-blockade, as excess norepinephrine is removed 65 in the analysis of genomic DNA. In certain aspects, a nucleic from the system. Norepinephrine is toxic to heart muscle and acid comprises a wild-type, a mutant, or a polymorphic in excess amounts triggers various cardiac disease pathways. nucleic acid. US 8,080,578 B2 23 24 The terms "B-adrenergic receptor polymorphisms or nucleic acid may encompass a double-stranded molecule or a “BAR’ polymorphisms, therefore, are terms of art and refer triple-stranded molecule that comprises one or more comple to polymorphisms in the nucleic acid oramino acid sequence mentary Strand(s) or “complement(s) of a particular of a B-adrenergic receptor gene or gene product. For refer sequence comprising a molecule. As used herein, a single ence purposes only, GenBank Accession No. J03019 stranded nucleic acid may be denoted by the prefix “ss', a (Gly389) and AF169007 (Arg389) (both of which are herein double stranded nucleic acid by the prefix “ds', and a triple incorporated by reference) are examples of Gly- and Arg-389 stranded nucleic acid by the prefix “ts.” forms of the B-adrenergic receptor gene sequence, respec In particular aspects, a nucleic acid encodes a protein, tively. polypeptide, or peptide. In certain embodiments, the present Also, the terms "C-adrenergic receptor polymorphisms 10 or "CAR' polymorphisms, therefore, are terms of art and invention concerns novel compositions comprising at least refer to polymorphisms in the nucleic acid or amino acid one proteinaceous molecule. As used herein, a “proteina sequence of a C2-adrenergic receptor gene or gene product. ceous molecule.” “proteinaceous composition.” “proteina For reference purposes only, GenBank Accession No. ceous compound,” “proteinaceous chain, or “proteinaceous NM00683 corresponds to wildtype (non-deletion) and 15 material' generally refers, but is not limited to, a protein of AF280400 corresponds to the deletion (both of which are greater than about 200 amino acids or the full length endog herein incorporated by reference). enous sequence translated from a gene; a polypeptide of For the purposes of identifying the location of a polymor greater than about 100 amino acids; and/or a peptide of from phism, the first nucleotide of the start codon of the coding about 3 to about 100 amino acids. All the “proteinaceous' region (the adenine of the ATG in a DNA molecule and the terms described above may be used interchangeably herein. adenine of the AUG in an RNA molecule) of the BAR gene 1. Preparation of Nucleic Acids or C-AR is considered nucleotide “1” and the numbers A nucleic acid may be made by any technique knownto one progress according along the coding sequence. Similarly, the of ordinary skill in the art, Such as for example, chemical first amino acid of the translated protein product (the synthesis, enzymatic production or biological production. methionine) is considered amino acid “1”. 25 Non-limiting examples of a synthetic nucleic acid (e.g., a The term “nucleic acid' is well known in the art. A "nucleic synthetic oligonucleotide), include a nucleic acid made by in acid as used herein will generally refer to a molecule (i.e., a vitro chemical synthesis using phosphotriester, phosphite or strand) of DNA or RNA comprising a nucleobase. A nucleo phosphoramidite chemistry and solid phase techniques such base includes, for example, a naturally occurring purine or as described in European Patent 266.032, incorporated herein pyrimidine base found in DNA (e.g., an adenine “A” a gua 30 by reference, or via deoxynucleoside H-phosphonate inter nine “G” a thymine “T” or a cytosine “C”) or RNA (e.g., an mediates as described by Froehler et al., 1986 and U.S. Pat. A, a G, anuracil"U” or a C). The term “nucleic acid” encom No. 5,705,629, each incorporated herein by reference. In the pass the terms "oligonucleotide' and “polynucleotide.” each methods of the present invention, one or more oligonucle as a subgenus of the term “nucleic acid.” The term "oligo otide may be used. Various different mechanisms of oligo nucleotide' refers to a molecule of between about 3 and about 35 nucleotide synthesis have been disclosed in for example, U.S. 100 nucleobases in length. The term “polynucleotide' refers Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959, to at least one molecule of greater than about 100 nucleobases 463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of in length. A "gene’ refers to coding sequence of a gene which is incorporated herein by reference. product, as well as introns and the promoter of the gene A non-limiting example of an enzymatically produced product. 40 nucleic acid include one produced by enzymes in amplifica In some embodiments, nucleic acids of the invention com tion reactions such as PCRTM (see for example, U.S. Pat. No. prise or are complementary to all or 5, 6,7,8,9, 10, 11, 12, 13, 4,683.202 and U.S. Pat. No. 4,682,195, each incorporated 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, herein by reference), or the synthesis of an oligonucleotide 31, 32,33, 34,35,36, 37,38, 39, 40, 41, 42,43, 44, 45, 46,47, described in U.S. Pat. No. 5,645,897, incorporated herein by 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62,63, 64, 45 reference. A non-limiting example of a biologically produced 65, 66,67,68, 69,70, 71,72, 73,74, 75,76, 77,78, 79,80, 81, nucleic acid includes a recombinant nucleic acid produced 82, 83, 84,85, 86, 87, 88, 89,90,91, 92,93,94, 95, 96, 97,98, (i.e., replicated) in a living cell. Such as a recombinant DNA 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, vector replicated in bacteria (see for example, Sambrooketal. 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 2001, incorporated herein by reference). 330, 340, 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 50 2. Purification of Nucleic Acids 450, 460, 470,480,490, 500,510,520, 530, 540, 550, 560, A nucleic acid may be purified on polyacrylamide gels, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, cesium chloride centrifugation gradients, chromatography 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, columns or by any other means known to one of ordinary skill 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, in the art (see for example, Sambrook et al., 2001, incorpo 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1165, 1200, 55 rated herein by reference). In some aspects, a nucleic acid is 1300, 1400, 1500 or more contiguous nucleotides, or any a pharmacologically acceptable nucleic acid. Pharmacologi range derivable therein, of the human BAR clNA sequence cally acceptable compositions are known to those of skill in with either a cytosine or guanine at position 1165 in the cDNA the art, and are described herein. sequence or of the CAR cDNA sequence with nucleotides In certain aspects, the present invention concerns a nucleic 964-975 present or absent. One of skill in the art knows how 60 acid that is an isolated nucleic acid. As used herein, the term to design and use primers and probes for hybridization and "isolated nucleic acid refers to a nucleic acid molecule (e.g., amplification, including the limits of homology needed to an RNA or DNA molecule) that has been isolated free of, or implement primers and probes. is otherwise free of the bulk of the total genomic and tran These definitions generally refer to a single-stranded mol scribed nucleic acids of one or more cells. In certain embodi ecule, but in specific embodiments will also encompass an 65 ments, "isolated nucleic acid refers to a nucleic acid that has additional strand that is partially, substantially or fully been isolated free of, or is otherwise free of, bulk of cellular complementary to the single-stranded molecule. Thus, a components or in vitro reaction components such as for US 8,080,578 B2 25 26 example, macromolecules such as lipids or proteins, Small from the individual a nucleic acid mixture comprising the two biological molecules, and the like. copies of the BAR gene, or a fragment thereof, that are 3. Nucleic Acid Segments present in the individual, and determining the identity of the In certain embodiments, the nucleic acid is a nucleic acid nucleotide pair at position 1165 in the BAR or determining segment. As used herein, the term “nucleic acid segment are whether there is a deletion of nucleotides 964-975 in the fragments of a nucleic acid, Such as, for a non-limiting CAR gene. Preferred polymorphisms and polymorphic example, those that encode only part of a faR gene locus or sites in a gene for a 3AR and CAR include the following in a BAR gene sequence, or part of the CAR gene locus or Table 1: gene sequence. Thus, a “nucleic acid segment may comprise any part of a gene sequence, including from about 2 nucle 10 otides to the full length gene including promoter regions to TABLE 1 the polyadenylation signal and any length that includes all the B-Adrenergic Receptor Polymorphism coding region. Nucleotide Amino Acid Various nucleic acid segments may be designed based on a Position Nucleotide Position Amino Acid Designations particular nucleic acid sequence, and may be of any length. 15 By assigning numeric values to a sequence, for example, the 1165 G or C 389 Gly or Arg Gly389, first residue is 1, the second residue is 2, etc., an algorithm Arg389 defining all nucleic acid segments can be created: C2-Adrenergic Receptor Polymorphism
in to n+y Nucleotide Amino Acid where n is an integer from 1 to the last number of the sequence Position Nucleotide Positions Amino Acid Designations and y is the length of the nucleic acid segment minus one, where n+y does not exceed the last number of the sequence. 964-975 deletion 322-32S deletion C-Del322-325 Thus, for a 10-mer, the nucleic acid segments correspond to bases 1 to 10, 2 to 11, 3 to 12... and so on. For a 15-mer, the 25 These polymorphisms in have been previously reported. nucleic acid segments correspond to bases 1 to 15, 2 to 16, 3 Wild-type BAR nucleotide sequences generally comprise a to 17 . . . and so on. For a 20-mer, the nucleic segments guanine at nucleotide 1165. Wild-type BAR protein correspond to bases 1 to 20, 2 to 21, 3 to 22 ... and so on. In sequences generally comprise a glycine at amino acid 389. certain embodiments, the nucleic acid segment may be a What is considered wild-type CAR nucleotide sequences probe or primer. As used herein, a “probe’ generally refers to 30 generally mean there is no deletion of nucleotides 964-975 a nucleic acid used in a detection method or composition. As and therefore no deletion of amino acids 322-325. used herein, a “primer generally refers to a nucleic acid used Those in the art will readily recognize that nucleic acid in an extension or amplification method or composition. molecules may be double-stranded molecules and that refer 4. Nucleic Acid Complements ence to a particular site on one strand refers, as well, to the The present invention also encompasses a nucleic acid that 35 corresponding site on a complementary strand. Thus, in is complementary to a nucleic acid. A nucleic acid is defining a polymorphic site, reference to an adenine, a thym “complement(s) or is “complementary to another nucleic ine (uridine), a cytosine, or a guanine at a particular site on the acid when it is capable of base-pairing with another nucleic plus (sense or coding) strand of a nucleic acid molecule is also acid according to the standard Watson-Crick, Hoogsteen or intended to include the thymine (uridine), adenine, guanine, reverse Hoogsteen binding complementarity rules. As used 40 or cytosine (respectively) at the corresponding site on a minus herein “another nucleic acid may refer to a separate mol (antisense or noncoding) strand of a complementary strand of ecule or a spatial separated sequence of the same molecule. In a nucleic acid molecule. Thus, reference may be made to preferred embodiments, a complement is a hybridization either strand and still comprise the same polymorphic site and probe or amplification primer for the detection of a nucleic an oligonucleotide may be designed to hybridize to either acid polymorphism. 45 Strand. Throughout the text, in identifying a polymorphic site, As used herein, the term “complementary' or “comple reference is made to the sense Strand, only for the purpose of ment also refers to a nucleic acid comprising a sequence of convenience. consecutive nucleobases or semiconsecutive nucleobases Typically, the nucleic acid mixture is isolated from a bio (e.g., one or more nucleobase moieties are not present in the logical sample taken from the individual. Such as a blood molecule) capable of hybridizing to another nucleic acid 50 sample or tissue sample using standard techniques such as strand or duplex even if less than all the nucleobases do not disclosed in Jones (1963) which is hereby incorporated by base pair with a counterpart nucleobase. However, in some reference. Suitable tissue samples include whole blood, diagnostic or detection embodiments, completely comple semen saliva, tears, urine, fecal material, Sweat, buccal, skin mentary nucleic acids are preferred. and hair. The nucleic acid mixture may be comprised of 5. Nucleic Acid Detection and Evaluation 55 genomic DNA, mRNA, or cDNA and, in the latter two cases, Genotyping was performed using methods exactly as pre the biological sample must be obtained from an organ in viously described in Small et al., (2002), which is incorpo which the BAR gene is expressed. Furthermore it will be rated herein by reference. It will be understood by the skilled understood by the skilled artisan that mRNA or cDNA prepa artisan that other standard techniques are available for geno rations would not be used to detect polymorphisms located in typing and any technique may be used with the present inven 60 introns or in 5' and 3' nontranscribed regions. If a BAR gene tion. General methods of nucleic acid detection methods are fragment is isolated, it must contain the polymorphic site(s) to provided below, followed by specific examples employed for be genotyped. the identification of polymorphisms, including single nucle The ability to predict a patient's response to a 3-agonist is otide polymorphisms (SNPs). useful for physicians in making decisions about how to treat The particular genotyping method used to determine the 65 a patient having heart failure. A patient whose genotype indi genotype of an individual in need of a B-blockertherapy is not cates the patient will probably respond well to the agonist part of the present invention, but in short involves isolating would be a better candidate for B-blocker therapy than a US 8,080,578 B2 27 28 patient who is likely to exhibit an intermediate response or no made of silicon, glass, plastic, paper and the like, which may response, and the physician would be able to determine with be formed, for example, into wells (as in 96-well plates), less trial and error which individuals should receive an alter slides, sheets, membranes, fibers, chips, dishes, and beads. native form of therapy. The solid support may be treated, coated or derivatized to In the genotyping methods used in the present invention, facilitate the immobilization of the allele-specific oligonucle the identity of a nucleotide (or nucleotide pair) at a polymor otide or target nucleic acid. phic site may be determined by amplifying a target region(s) The genotype for one or more polymorphic sites in the containing the polymorphic site(s) directly from one or both BAR gene of an individual may also be determined by copies of the faR gene and/or CAR gene present in the hybridization of one or both copies of the gene, or a fragment individual and the sequence of the amplified region(s) deter 10 thereof, to nucleic acid arrays and Subarrays such as described mined by conventional methods. It will be readily appreciated in WO95/11995. The arrays would contain a battery of allele by the skilled artisan that only one nucleotide will be detected specific oligonucleotides representing each of the polymor at a polymorphic site in individuals who are homozygous at phic sites to be included in the genotype or haplotype. that site, while two different nucleotides will be detected if the The identity of polymorphisms may also be determined individual is heterozygous for that site. The polymorphism 15 using a mismatch detection technique, including but not lim may be identified directly, known as positive-type identifica ited to the RNase protection method using riboprobes (Winter tion, or by inference, referred to as negative-type identifica et al., 1985; Meyers et al., 1985) and proteins which recognize tion. For example, where a SNP is known to be guanine and nucleotide mismatches, such as the E. coli mutS protein cytosine in a reference population, a site may be positively (Modrich, 1991). Alternatively, variant alleles can be identi determined to be either guanine or cytosine for an individual fied by single strand conformation polymorphism (SSCP) homozygous at that site, or both guanine and cytosine, if the analysis (Orita et al., 1989: Humphries, et al., 1996) or dena individual is heterozygous at that site. Alternatively, the site turing gradient gel electrophoresis (DGGE) (Wartell et al., may be negatively determined to be not guanine (and thus 1990; Sheffield et al., 1989). cytosine? cytosine) or not cytosine (and thus guanine/gua A polymerase-mediated primer extension method may nine). 25 also be used to identify the polymorphism(s). Several such The target region(s) may be amplified using any oligo methods have been described in the patent and scientific nucleotide-directed amplification method, including but not literature. Extended primers containing a polymorphism may limited to polymerase chain reaction (PCR) (U.S. Pat. No. be detected by mass spectrometry as described in U.S. Pat. 4.965, 188), ligase chain reaction (LCR) (Barany et al., 1991; No. 5,605,798. An other primer extension method is allele WO90/01069), and oligonucleotide ligation assay (OLA) 30 specific PCR (Ruano et al., 1989); Ruano et al., 1991; WO (Landegren et al., 1988). Oligonucleotides useful as primers 93/224.56: Turki et al., 1995). or probes in such methods should specifically hybridize to a Polymorphic variation at nucleotide position 1165 of the region of the nucleic acid that contains or is adjacent to the human fBAR gene can also be detected using differential polymorphic site. Typically, the oligonucleotides are between digestion of DNA by certain restriction enzymes (Smallet al., 10 and 35 nucleotides in length and preferably, between 15 35 2002) or by any other method that identifies the nucleotide at and 30 nucleotides in length. Most preferably, the oligonucle position 1165 of the BAR gene. otides are 20 to 25 nucleotides long. The exact length of the a. Hybridization oligonucleotide will depend on many factors that are rou The use of a probe or primer of between 7, 8, 9, 10, 11, 12, tinely considered and practiced by the skilled artisan. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 70, 80, Other known nucleic acid amplification procedures may be 40 90, or 100 nucleotides, preferably between 17 and 100 nucle used to amplify the target region including transcription otides in length, or in some aspects of the invention up to 1-2 based amplification systems (U.S. Pat. No. 5,130.238; EP kilobases or more in length, allows the formation of a duplex 329,822; U.S. Pat. No. 5,169,766, WO89/06700) and isother molecule that is both stable and selective. Molecules having mal methods (Walker et al., 1992). complementary sequences over contiguous stretches greater A polymorphism in the target region may also be assayed 45 than 20 bases in length are generally preferred, to increase before or after amplification using one of several hybridiza stability and/or selectivity of the hybrid molecules obtained. tion-based methods known in the art. Typically, allele-spe One will generally prefer to design nucleic acid molecules for cific oligonucleotides are utilized in performing Such meth hybridization having one or more complementary sequences ods. The allele-specific oligonucleotides may be used as of 20 to 30 nucleotides, or even longer where desired. Such differently labeled probe pairs, with one member of the pair 50 fragments may be readily prepared, for example, by directly showing a perfect match to one variant of a target sequence synthesizing the fragment by chemical means or by introduc and the other member showing a perfect match to a different ing selected sequences into recombinant vectors for recom variant. In some embodiments, more than one polymorphic binant production. site may be detected at once using a set of allele-specific Accordingly, the nucleotide sequences of the invention oligonucleotides or oligonucleotide pairs. 55 may be used for their ability to selectively form duplex mol Hybridization of an allele-specific oligonucleotide to a ecules with complementary stretches of DNAs and/or RNAs target polynucleotide may be performed with both entities in or to provide primers for amplification of DNA or RNA from solution, or such hybridization may be performed when either samples. Depending on the application envisioned, one the oligonucleotide or the target polynucleotide is covalently would desire to employ varying conditions of hybridization to or noncovalently affixed to a solid Support. Attachment may 60 achieve varying degrees of selectivity of the probe or primers be mediated, for example, by antibody-antigen interactions, for the target sequence. poly-L-Lys, streptavidin oravidin-biotin, Salt bridges, hydro For applications requiring high selectivity, one will typi phobic interactions, chemical linkages, UV cross-linking cally desire to employ relatively high Stringency conditions to baking, etc. Allele-specific oligonucleotides may be synthe form the hybrids. For example, relatively low salt and/or high sized directly on the solid support or attached to the solid 65 temperature conditions, such as provided by about 0.02 M to Support Subsequent to synthesis. Solid-supports suitable for about 0.10 M. NaCl attemperatures of about 50° C. to about use in detection methods of the invention include substrates 70° C. Such high stringency conditions tolerate little, if any, US 8,080,578 B2 29 30 mismatch between the probe or primers and the template or methods of hybridization that may be used in the practice of target Strand and would be particularly Suitable for isolating the present invention are disclosed in U.S. Pat. Nos. 5,849, specific genes or for detecting a specific polymorphism. It is 481, 5,849,486 and 5,851,772. The relevant portions of these generally appreciated that conditions can be rendered more and other references identified in this section of the Specifi stringent by the addition of increasing amounts of formamide. 5 cation are incorporated herein by reference. For example, under highly stringent conditions, hybridization b. Amplification of Nucleic Acids to filter-bound DNA may be carried out in 0.5 M NaHPO, Nucleic acids used as a template for amplification may be 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C., and isolated from cells, tissues or other samples according to washing in 0.1xSSC/0.1% SDS at 68° C. (Ausubel et al., standard methodologies (Sambrook et al., 2001). In certain 1989). 10 embodiments, analysis is performed on whole cell or tissue Conditions may be rendered less stringent by increasing homogenates or biological fluid samples with or without Sub salt concentration and/or decreasing temperature. For stantial purification of the template nucleic acid. The nucleic example, a medium stringency condition could be provided acid may be genomic DNA or fractionated or whole cell by about 0.1 to 0.25 MNaCl attemperatures of about 37° C. RNA. Where RNA is used, it may be desired to first convert to about 55° C., while a low stringency condition could be 15 the RNA to a complementary DNA. provided by about 0.15 M to about 0.9 Msalt, attemperatures The term “primer, as used herein, is meant to encompass ranging from about 20°C. to about 55°C. Under low stringent any nucleic acid that is capable of priming the synthesis of a conditions, such as moderately stringent conditions the wash nascent nucleic acid in a template-dependent process. Typi ing may be carried out for example in 0.2xSSC/0.1% SDS at cally, primers are oligonucleotides from ten to twenty and/or 42°C. (Ausubel et al., 1989). Hybridization conditions can be thirty base pairs in length, but longer sequences can be readily manipulated depending on the desired results. employed. Primers may be provided in double-stranded and/ In other embodiments, hybridization may be achieved or single-stranded form, although the single-stranded form is under conditions of, for example, 50 mM Tris-HCl (pH 8.3), preferred. 75 mM KC1, 3 mM MgCl, 1.0 mM dithiothreitol, at tem Pairs of primers designed to selectively hybridize to peratures between approximately 20° C. to about 37° C. 25 nucleic acids corresponding to the BAR gene locus, or vari Other hybridization conditions utilized could include ants thereof, and fragments thereof are contacted with the approximately 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 template nucleic acid under conditions that permit selective mM MgCl2, attemperatures ranging from approximately 40° hybridization. Depending upon the desired application, high C. to about 72° C. stringency hybridization conditions may be selected that will In certain embodiments, it will be advantageous to employ 30 only allow hybridization to sequences that are completely nucleic acids of defined sequences of the present invention in complementary to the primers. In other embodiments, combination with an appropriate means, such as a label, for hybridization may occur under reduced stringency to allow determining hybridization. A wide variety of appropriate for amplification of nucleic acids that contain one or more indicator means are known in the art, including fluorescent, mismatches with the primer sequences. Once hybridized, the radioactive, enzymatic or other ligands. Such as avidin/biotin, 35 template-primer complex is contacted with one or more which are capable of being detected. In preferred embodi enzymes that facilitate template-dependent nucleic acid syn ments, one may desire to employ a fluorescent label or an thesis. Multiple rounds of amplification, also referred to as enzyme tag such as urease, alkaline phosphatase or peroxi “cycles are conducted until a sufficient amount of amplifi dase, instead of radioactive or other environmentally unde cation product is produced. sirable reagents. In the case of enzyme tags, colorimetric 40 The amplification product may be detected, analyzed or indicator Substrates are known that can be employed to pro quantified. In certain applications, the detection may be per vide a detection means that is visibly or spectrophotometri formed by visual means. In certain applications, the detection cally detectable, to identify specific hybridization with may involve indirect identification of the product via chemi complementary nucleic acid containing samples. In other luminescence, radioactive Scintigraphy of incorporated aspects, a particular nuclease cleavage site may be present 45 radiolabel or fluorescent label or even via a system using and detection of a particular nucleotide sequence can be electrical and/or thermal impulse signals (Affymax technol determined by the presence or absence of nucleic acid cleav ogy: Bellus, 1994). age. A number oftemplate dependent processes are available to In general, it is envisioned that the probes or primers amplify the oligonucleotide sequences present in a given described herein will be useful as reagents in solution hybrid 50 template sample. One of the best known amplification meth ization, as in PCR, for detection of expression or genotype of ods is the polymerase chain reaction (referred to as PCRTM) corresponding genes, as well as in embodiments employing a which is described in detail in U.S. Pat. Nos. 4,683, 195, Solid phase. In embodiments involving a solid phase, the test 4,683.202 and 4,800,159, and in Innis et al., 1988, each of DNA (or RNA) is adsorbed or otherwise affixed to a selected which is incorporated herein by reference in their entirety. matrix or Surface. This fixed, single-stranded nucleic acid is 55 Another method for amplification is ligase chain reaction then subjected to hybridization with selected probes under (“LCR), disclosed in European Application No. 320 308, desired conditions. The conditions selected will depend on incorporated herein by reference in its entirety. U.S. Pat. No. the particular circumstances (depending, for example, on the 4,883,750 describes a method similar to LCR for binding G+C content, type of target nucleic acid, Source of nucleic probe pairs to a target sequence. A method based on PCRTM acid, size of hybridization probe, etc.). Optimization of 60 and oligonucleotide ligase assay (OLA) (described in further hybridization conditions for the particular application of detail below), disclosed in U.S. Pat. No. 5,912,148, may also interest is well known to those of skill in the art. After washing be used. of the hybridized molecules to remove non-specifically Alternative methods for amplification of target nucleic acid bound probe molecules, hybridization is detected, and/or sequences that may be used in the practice of the present quantified, by determining the amount of bound label. Rep 65 invention are disclosed in U.S. Pat. Nos. 5,843,650, 5,846, resentative solid phase hybridization methods are disclosed in 709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906, US 8,080,578 B2 31 32 5,932,451, 5,935,825, 5,939,291 and 5,942,391, Great Brit In particular embodiments, detection is by Southern blot ain Application 2 202 328, and in PCT Application PCT/ ting and hybridization with a labeled probe. The techniques US89/01025, each of which is incorporated herein by refer involved in Southern blotting are well known to those of skill ence in its entirety. Qbeta Replicase, described in PCT in the art (see Sambrook et al., 2001). One example of the Application PCT/US87/00880, may also be used as an ampli foregoing is described in U.S. Pat. No. 5.279,721, incorpo fication method in the present invention. rated by reference herein, which discloses an apparatus and An isothermal amplification method, in which restriction method for the automated electrophoresis and transfer of endonucleases and ligases are used to achieve the amplifica nucleic acids. The apparatus permits electrophoresis and blot tion of target molecules that contain nucleotide 5'-alpha ting without external manipulation of the gel and is ideally thio-triphosphates in one strand of a restriction site may also 10 Suited to carrying out methods according to the present inven be useful in the amplification of nucleic acids in the present tion. invention (Walker et al., 1992). Strand Displacement Ampli Other methods of nucleic acid detection that may be used in fication (SDA), disclosed in U.S. Pat. No. 5,916,779, is the practice of the instant invention are disclosed in U.S. Pat. another method of carrying out isothermal amplification of Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717, nucleic acids which involves multiple rounds of strand dis 15 5,846,726, 5,846,729, 5,849.487, 5,853,990, 5,853,992, placement and synthesis, i.e., nick translation 5,853,993, 5,856,092, 5,861,244, 5,863,732, 5,863,753, Other nucleic acid amplification procedures include tran 5,866,331, 5,905,024, 5,910,407, 5,912,124, 5,912,145, Scription-based amplification systems (TAS), including 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227, nucleic acid sequence based amplification (NASBA) and 3SR 5,932,413 and 5.935,791, each of which is incorporated (Kwoh et al., 1989; PCT Application WO 88/10315, incor herein by reference. porated herein by reference in their entirety). European d. Other Assays Application 329 822 disclose a nucleic acid amplification Other methods for genetic screening may be used within process involving cyclically synthesizing single-stranded the scope of the present invention, for example, to detect RNA (“ssRNA), ssDNA, and double-stranded DNA (ds mutations in genomic DNA, cDNA and/or RNA samples. DNA), which may be used in accordance with the present 25 Methods used to detect point mutations include denaturing invention. gradient gel electrophoresis (“DGGE), restriction fragment PCT Application WO 89/06700 (incorporated herein by length polymorphism analysis (“RFLP), chemical or enzy reference in its entirety) disclose a nucleic acid sequence matic cleavage methods, direct sequencing of target regions amplification scheme based on the hybridization of a pro amplified by PCRTM (see above), single-strand conformation moter region/primer sequence to a target single-stranded 30 polymorphism analysis (“SSCP) and other methods well DNA (“ssDNA) followed by transcription of many RNA known in the art. copies of the sequence. This scheme is not cyclic, i.e., new One method of screening for point mutations is based on templates are not produced from the resultant RNA tran RNase cleavage of base pair mismatches in RNA/DNA or scripts. Other amplification methods include “RACE and RNA/RNA heteroduplexes. As used herein, the term “mis “one-sided PCR" (Frohman, 1990; Ohara et al., 1989). 35 match’ is defined as a region of one or more unpaired or c. Detection of Nucleic Acids mispaired nucleotides in a double-stranded RNA/RNA, Following any amplification, it may be desirable to sepa RNA/DNA or DNA/DNA molecule. This definition thus rate the amplification product from the template and/or the includes mismatches due to insertion/deletion mutations, as excess primer. In one embodiment, amplification products are well as single or multiple base point mutations. separated by agarose, agarose-acrylamide or polyacrylamide 40 U.S. Pat. No. 4,946,773 describes an RNase A mismatch gel electrophoresis using standard methods (Sambrook et al., cleavage assay that involves annealing single-stranded DNA 2001). Separated amplification products may be cut out and or RNA test samples to an RNA probe, and subsequent treat eluted from the gel for further manipulation. Using low melt ment of the nucleic acid duplexes with RNase A. For the ing point agarose gels, the separated band may be removed by detection of mismatches, the single-stranded products of the heating the gel, followed by extraction of the nucleic acid. 45 RNase A treatment, electrophoretically separated according Separation of nucleic acids may also be effected by spin to size, are compared to similarly treated control duplexes. columns and/or chromatographic techniques known in art. Samples containing Smaller fragments (cleavage products) There are many kinds of chromatography which may be used not seen in the control duplex are scored as positive. in the practice of the present invention, including adsorption, Other investigators have described the use of RNase I in partition, ion-exchange, hydroxylapatite, molecular sieve, 50 mismatch assays. The use of RNase I for mismatch detection reverse-phase, column, paper, thin-layer, and gas chromatog is described in literature from Promega Biotech. Promega raphy as well as HPLC. markets a kit containing RNase I that is reported to cleave In certain embodiments, the amplification products are three out of four known mismatches. Others have described visualized, with or without separation. A typical visualization using the MutS protein or other DNA-repair enzymes for method involves staining of a gel with ethidium bromide and 55 detection of single-base mismatches. visualization of bands under UV light. Alternatively, if the Alternative methods for detection of deletion, insertion or amplification products are integrally labeled with radio- or substitution mutations that may be used in the practice of the fluorometrically-labeled nucleotides, the separated amplifi present invention are disclosed in U.S. Pat. Nos. 5,849.483. cation products can be exposed to X-ray film or visualized 5,851,770, 5,866,337, 5,925,525 and 5,928,870, each of under the appropriate excitatory spectra. 60 which is incorporated herein by reference in its entirety. In one embodiment, following separation of amplification e. Specific Examples of Polymorphism Nucleic Acid products, a labeled nucleic acid probe is brought into contact Screening Methods with the amplified marker sequence. The probe preferably is Spontaneous mutations that arise during the course of evo conjugated to a chromophore but may be radiolabeled. In lution in the genomes of organisms are often not immediately another embodiment, the probe is conjugated to a binding 65 transmitted throughout all of the members of the species, partner, Such as an antibody or biotin, or another binding thereby creating polymorphic alleles that co-exist in the spe partner carrying a detectable moiety. cies populations. Often polymorphisms are the cause of US 8,080,578 B2 33 34 genetic diseases. Several classes of polymorphisms have been iii. MicroSequencing Methods identified. For example, variable nucleotide type polymor Several other primer-guided nucleotide incorporation pro phisms (VNTRS), arise from spontaneous tandem duplica cedures for assaying polymorphic sites in DNA have been tions of di- or trinucleotide repeated motifs of nucleotides. If described (Komher et al., 1989: Sokolov, 1990; Syvanen 5 1990; Kuppuswamy et al., 1991; Prezant et al., 1992; Ugoz Such variations alter the lengths of DNA fragments generated Zollet al., 1992; Nyrenet al., 1993). These methods rely on by restriction endonuclease cleavage, the variations are the incorporation of labeled deoxynucleotides to discriminate referred to as restriction fragment length polymorphisms between bases at a polymorphic site. As the signal is propor (RFLPs). RFLPs are been widely used in human and animal tional to the number of deoxynucleotides incorporated, poly genetic analyses. morphisms that occur in runs of the same nucleotide result in Another class of polymorphisms are generated by the 10 a signal that is proportional to the length of the run (Syvanen replacement of a single nucleotide. Such single nucleotide et al., 1990). polymorphisms (SNPs) rarely result in changes in a restric iv. Extension in Solution tion endonuclease site. Thus, SNPs are rarely detectable French Patent 2,650,840 and PCT Application WO91/ restriction fragment length analysis. SNPs are the most com 02087 discuss a solution-based method for determining the 15 identity of the nucleotide of a polymorphic site. According to mon genetic variations and occur once every 100 to 300 bases these methods, a primer complementary to allelic sequences and several SNP mutations have been found that affect a immediately 3'- to a polymorphic site is used. The identity of single nucleotide in a protein-encoding gene in a manner the nucleotide of that site is determined using labeled Sufficient to actually cause agenetic disease. SNP diseases are dideoxynucleotide derivatives which are incorporated at the exemplified by hemophilia, sickle-cell anemia, hereditary end of the primer if complementary to the nucleotide of the hemochromatosis, late-onset alzheimer disease etc. polymorphic site. Several methods have been developed to screen polymor V. Genetic Bit Analysis or Solid-Phase Extension phisms and some examples are listed below. The reference of PCT Application WO92/15712 describes a method that Kwok and Chen (2003) and Kwok (2001) provide overviews uses mixtures of labeled terminators and a primer that is of some of these methods; both of these references are spe 25 complementary to the sequence 3' to a polymorphic site. The cifically incorporated by reference. labeled terminator that is incorporated is complementary to SNPs relating to ABCC2 can be characterized by the use of the nucleotide present in the polymorphic site of the target any of these methods or suitable modification thereof. Such molecule being evaluated and is thus identified. Here the methods include the direct or indirect sequencing of the site, primer or the target molecule is immobilized to a Solid phase. the use of restriction enzymes where the respective alleles of 30 vi. Oligonucleotide Ligation Assay (OLA) the site create or destroy a restriction site, the use of allele This is another solid phase method that uses different meth specific hybridization probes, the use of antibodies that are odology (Landegren et al., 1988). Two oligonucleotides, specific for the proteins encoded by the differentalleles of the capable of hybridizing to abutting sequences of a single polymorphism, or any other biochemical interpretation. strand of a target DNA are used. One of these oligonucle i. DNA Sequencing 35 otides is biotinylated while the other is detectably labeled. If The most commonly used method of characterizing a poly the precise complementary sequence is found in a target morphism is direct DNA sequencing of the genetic locus that molecule, the oligonucleotides will hybridize such that their flanks and includes the polymorphism. Such analysis can be termini abut, and create a ligation Substrate. Ligation permits accomplished using either the "dideoxy-mediated chain ter the recovery of the labeled oligonucleotide by using avidin. mination method, also known as the “Sanger Method 40 Other nucleic acid detection assays, based on this method, (Sanger et al., 1975) or the “chemical degradation method.” combined with PCR have also been described (Nickerson et also known as the “Maxam-Gilbert method’ (Maxam et al., al., 1990). Here PCR is used to achieve the exponential ampli 1977). Sequencing in combination with genomic sequence fication of target DNA, which is then detected using the OLA. specific amplification technologies, such as the polymerase vii. Ligase/Polymerase-Mediated Genetic Bit Analysis chain reaction may be utilized to facilitate the recovery of the 45 U.S. Pat. No. 5,952,174 describes a method that also desired genes (Mullis et al., 1986: European Patent Applica involves two primers capable of hybridizing to abutting tion 50,424; European Patent Application. 84.796, European sequences of a target molecule. The hybridized product is Patent Application 258,017, European Patent Application. formed on a solid support to which the target is immobilized. 237,362: European Patent Application. 201,184; U.S. Pat. Here the hybridization occurs such that the primers are sepa Nos. 4,683.202; 4,582,788; and 4,683,194), all of the above 50 rated from one another by a space of a single nucleotide. incorporated herein by reference. Incubating this hybridized product in the presence of a poly ii. Exonuclease Resistance merase, a ligase, and a nucleoside triphosphate mixture con Other methods that can be employed to determine the taining at least one deoxynucleoside triphosphate allows the identity of a nucleotide present at a polymorphic site utilize a ligation of any pair of abutting hybridized oligonucleotides. specialized exonuclease-resistant nucleotide derivative (U.S. 55 Addition of a ligase results in two events required to generate Pat. No. 4,656,127). A primer complementary to an allelic a signal, extension and ligation. This provides a higher speci sequence immediately 3'- to the polymorphic site is hybrid ficity and lower “noise than methods using either extension ized to the DNA under investigation. If the polymorphic site or ligation alone and unlike the polymerase-based assays, this on the DNA contains a nucleotide that is complementary to method enhances the specificity of the polymerase step by the particular exonucleotide-resistant nucleotide derivative 60 combining it with a second hybridization and a ligation step present, then that derivative will be incorporated by a poly for a signal to be attached to the Solid phase. merase onto the end of the hybridized primer. Such incorpo viii. Invasive Cleavage Reactions ration makes the primer resistant to exonuclease cleavage and Invasive cleavage reactions can be used to evaluate cellular thereby permits its detection. As the identity of the exonucle DNA for a particular polymorphism. A technology called otide-resistant derivative is known one can determine the 65 INVADER(R) employs such reactions (e.g., de Arruda et al., specific nucleotide present in the polymorphic site of the 2002; Stevens et al., 2003, which are incorporated by refer DNA. ence). Generally, there are three nucleic acid molecules: 1) an US 8,080,578 B2 35 36 oligonucleotide upstream of the target site ("upstream In a method called CGAP-GAI (DEMIGLACE), sequence oligo'), 2) a probe oligonucleotide covering the target site and alignment data (from a PHRAPace file), quality scores (“probe'), and 3) a single-stranded DNA with the target site for the sequence base calls (from PHRED quality files), dis (“target”). The upstream oligo and probe do not overlap but tance information (from PHYLIP dnadist and neighbour pro they contain contiguous sequences. The probe contains a grams) and base-calling data (from PHRED -d switch) are donor fluorophore, such as fluoroscein, and an acceptor dye, loaded into memory. Sequences are aligned and examined for such as Dabcyl. The nucleotide at the 3' terminal end of the each vertical chunk ('slice) of the resulting assembly for upstream oligo overlaps (“invades”) the first base pair of a disagreement. Any such slice is considered a candidate SNP probe-target duplex. Then the probe is cleaved by a structure (DEMIGLACE). A number of filters are used by specific 5' nuclease causing separation of the fluorophore/ 10 DEMIGLACE to eliminate slices that are not likely to repre sent true polymorphisms. These include filters that: (i) quencher pair, which increases the amount of fluorescence exclude sequences in any given slice from SNP consideration that can be detected. See Lu et al., 2004. where neighboring sequence quality Scores drop 40% or In some cases, the assay is conducted on a solid-surface or more; (ii) exclude calls in which peak amplitude is below the in an array format. 15 fifteenth percentile of all base calls for that nucleotide type: ix. Other Methods To Detect SNPs (iii) disqualify regions of a sequence having a high number of Several other specific methods for polymorphism detec disagreements with the consensus from participating in SNP tion and identification are presented below and may be used calculations; (iv) removed from consideration any base call as Such or with Suitable modifications in conjunction with with an alternative call in which the peak takes up 25% or identifying polymorphisms of the BAR gene in the present more of the area of the called peak; (V) exclude variations that invention. Several other methods are also described on the occur in only one read direction. PHRED quality scores were SNP web site of the NCBI on the World WideWeb at ncbi.n- converted into probability-of-error values for each nucleotide lm.nih.gov/SNP, incorporated herein by reference. in the slice. Standard Baysian methods are used to calculate In a particular embodiment, extended haplotypes may be the posterior probability that there is evidence of nucleotide determined at any given locus in a population, which allows 25 heterogeneity at a given location. one to identify exactly which SNPs will be redundant and In a method called CU-RDF (RESEQ), PCR amplification which will be essential in association studies. The latter is is performed from DNA isolated from blood using specific referred to as haplotype tag SNPs (htSNPs), markers that primers for each SNP, and after typical cleanup protocols to capture the haplotypes of a gene or a region of linkage dis remove unused primers and free nucleotides, direct sequenc equilibrium. See Johnson et al. (2001) and Ke and Cardon 30 ing using the same or nested primers. (2003), each of which is incorporated herein by reference, for In a method called DEBNICK (METHOD-B), a compara exemplary methods. tive analysis of clustered EST sequences is performed and The VDA-assay utilizes PCR amplification of genomic confirmed by fluorescent-based DNA sequencing. In a related segments by long PCR methods using TakaRa LA Taq method, called DEBNICK (METHOD-C), comparative reagents and other standard reaction conditions. The long 35 analysis of clustered EST sequences with phredduality >20 at amplification can amplify DNA sizes of about 2,000-12,000 the site of the mismatch, average phred quality >=20 over 5 bp. Hybridization of products to variant detector array (VDA) bases 5'-FLANK and 3' to the SNP, no mismatches in 5 bases can be performed by a Affymetrix High Throughput Screen 5' and 3' to the SNP, at least two occurrences of each allele is ing Center and analyzed with computerized software. performed and confirmed by examining traces. A method called Chip Assay uses PCR amplification of 40 In a method identified by ERO (RESEW), new primers sets genomic segments by standard or long PCR protocols. are designed for electronically published STSs and used to Hybridization products are analyzed by VDA. Halushka et al. amplify DNA from 10 different mouse strains. The amplifi (1999), incorporated herein by reference. SNPs are generally cation product from each strain is then gel purified and classified as “Certain' or “Likely based on computer analy sequenced using a standard dideoxy, cycle sequencing tech sis of hybridization patterns. By comparison to alternative 45 nique with P-labeled terminators. All the ddATP terminated detection methods such as nucleotide sequencing, “Certain' reactions are then loaded in adjacent lanes of a sequencing gel SNPs have been confirmed 100% of the time; and “Likely” followed by all of the ddGTP reactions and so on. SNPs are SNPs have been confirmed 73% of the time by this method. identified by visually scanning the radiographs. Other methods simply involve PCR amplification follow In another method identified as ERO (RESEQ-HT), new ing digestion with the relevant restriction enzyme. Yet others 50 primers sets are designed for electronically published murine involve sequencing of purified PCR products from known DNA sequences and used to amplify DNA from 10 different genomic regions. mouse strains. The amplification product from each strain is In yet another method, individual exons or overlapping prepared for sequencing by treating with Exonuclease I and fragments of large exons are PCR-amplified. Primers are Shrimp Alkaline Phosphatase. Sequencing is performed designed from published or database sequences and PCR 55 using ABI Prism Big Dye Terminator Ready Reaction Kit amplification of genomic DNA is performed using the fol (Perkin-Elmer) and sequence samples are run on the 3700 lowing conditions: 200 ng DNA template, 0.5 LM each DNA Analyzer (96 Capillary Sequencer). primer, 80 uM each of dCTP, dATP, dTTP and dGTP, 5% FGU-CBT (SCA2-SNP) identifies a method where the formamide, 1.5 mMMgCl2, 0.5 U of Taq polymerase and 0.1 region containing the SNP were PCR amplified using the volume of the Taq buffer. Thermal cycling is performed and 60 primers SCA2-FP3 and SCA2-RP3. Approximately 100 ng resulting PCR-products are analyzed by PCR-single strand of genomic DNA is amplified in a 50 ml reaction volume conformation polymorphism (PCR-SSCP) analysis, under a containing a final concentration of 5 mM Tris, 25 mM KC1, variety of conditions, e.g. 5 or 10% polyacrylamide gel with 0.75 mM MgCl, 0.05% gelatin, 20 umol of each primer and 15% urea, with or without 5% glycerol. Electrophoresis is 0.5 U of Taq DNA polymerase. Samples are denatured, performed overnight. PCR-products that show mobility shifts 65 annealed and extended and the PCR product is purified from are reamplified and sequenced to identify nucleotide varia a band cut out of the agarose gel using, for example, the tion. QIAquick gel extraction kit (Qiagen) and is sequenced using US 8,080,578 B2 37 38 dye terminator chemistry on an ABI Prism 377 automated tion as opposed to sequencing error. This process requires the DNA sequencer with the PCR primers. presence of base quality values for both sequences. High In a method identified as JBLACK (SEQ/RESTRICT), two scoring candidates are extracted. The search is restricted to independent PCR reactions are performed with genomic Substitution-type single base pair variations. Confidence DNA. Products from the first reaction are analyzed by score of candidate SNP is computed by the POLYBAYES sequencing, indicating a unique FspI restriction site. The software. mutation is confirmed in the product of the second PCR In method identified by KWOK (TaqMan assay), the Taq reaction by digesting with Fsp I. Man assay is used to determine genotypes for 90 random In a method described as KWOK(1), SNPs are identified individuals. In method identified by KYUGEN(Q1), DNA by comparing high quality genomic sequence data from four 10 samples of indicated populations are pooled and analyzed by randomly chosen individuals by direct DNA sequencing of PLACE-SSCP. Peak heights of each allele in the pooled PCR products with dye-terminator chemistry (see Kwok et analysis are corrected by those in a heterozygote, and are al., 1996). In a related method identified as KWOK(2) SNPs subsequently used for calculation of allele frequencies. Allele are identified by comparing high quality genomic sequence frequencies higher than 10% are reliably quantified by this data from overlapping large-insert clones such as bacterial 15 method. Allele frequency=0 (zero) means that the allele was artificial chromosomes (BACs) or P1-based artificial chro found among individuals, but the corresponding peak is not mosomes (PACs). An STS containing this SNP is then devel seen in the examination of pool. Allele frequency–0-0.1 indi oped and the existence of the SNP in various populations is cates that minor alleles are detected in the pool but the peaks confirmed by pooled DNA sequencing (see Taillon-Miller et are too low to reliably quantify. al., 1998). In another similar method called KWOK(3), SNPs In yet another method identified as KYUGEN (Method 1), are identified by comparing high quality genomic sequence PCR products are post-labeled with fluorescent dyes and data from overlapping large-insert clones BACs or PACs. The analyzed by an automated capillary electrophoresis system SNPs found by this approach represent DNA sequence varia under SSCP conditions (PLACE-SSCP). Four or more indi tions between the two donor chromosomes but the allele vidual DNAs are analyzed with or without two pooled DNA frequencies in the general population have not yet been deter 25 (Japanese pool and CEPH parents pool) in a series of experi mined. In method KWOK(5), SNPs are identified by com ments. Alleles are identified by visual inspection. Individual paring high quality genomic sequence data from a homozy DNAs with different genotypes are sequenced and SNPs gous DNA sample and one or more pooled DNA samples by identified. Allele frequencies are estimated from peak heights direct DNA sequencing of PCR products with dye-terminator in the pooled samples after correction of signal bias using chemistry. The STSs used are developed from sequence data 30 peak heights in heterozygotes. For the PCR primers are found in publicly available databases. Specifically, these tagged to have 5'-ATT or 5'-GTT at their ends for post-label STSs are amplified by PCR against a complete hydatidiform ing of both strands. Samples of DNA (10 ngful) are amplified mole (CHM) that has been shown to be homozygous at all loci in reaction mixtures containing the buffer (10 mM Tris-HCl, and a pool of DNA samples from 80 CEPH parents (see Kwok pH 8.3 or 9.3, 50 mM KC1, 2.0 mMMgCl), 0.25uMofeach et al., 1994). 35 primer, 200uMofeach dNTP and 0.025 units/ul of Taq DNA In another such method, KWOK (OverlapSnpDetection polymerase premixed with anti-Taq antibody. The two WithPolyl)ayes), SNPs are discovered by automated com strands of PCR products are differentially labeled with nucle puter analysis of overlapping regions of large-insert human otides modified with R110 and R6G by an exchange reaction genomic clone sequences. For data acquisition, clone of Klenow fragment of DNA polymerase I. The reaction is sequences are obtained directly from large-scale sequencing 40 stopped by adding EDTA, and unincorporated nucleotides are centers. This is necessary because base quality sequences are dephosphorylated by adding calf intestinal alkaline phos not present/available through GenBank. Raw data processing phatase. For the SSCP: an aliquot of fluorescently labeled involves analyzed of clone sequences and accompanying PCR products and TAMRA-labeled internal markers are base quality information for consistency. Finished (base per added to deionized formamide, and denatured. Electrophore fect, error rate lower than 1 in 10,000 bp) sequences with no 45 sis is performed in a capillary using an ABI Prism 310 Genetic associated base quality sequences are assigned a uniform Analyzer. Genescan softwares (P-E Biosystems) are used for base quality value of 40 (1 in 10,000 bp error rate). Draft data collection and data processing. DNA of individuals (two sequences without base quality values are rejected. Processed to eleven) including those who showed different genotypes on sequences are entered into a local database. A version of each SSCP are subjected for direct sequencing using big-dye ter sequence with known human repeats masked is also stored. 50 minator chemistry, on ABI Prism 310 sequencers. Multiple Repeat masking is performed with the program sequence trace files obtained from ABI Prism 310 are pro “MASKERAID.” Overlap detection: Putative overlaps are cessed and aligned by Phred/Phrap and viewed using Consed detected with the program “WUBLAST.” Several filtering viewer. SNPs are identified by PolyPhred software and visual steps followed in order to eliminate false overlap detection inspection. results, i.e. similarities between a pair of clone sequences that 55 In yet another method identified as KYUGEN (Method2), arise due to sequence duplication as opposed to true overlap. individuals with different genotypes are searched by denatur Total length of overlap, overall percent similarity, number of ing HPLC (DHPLC) or PLACE-SSCP (Inazuka et al., 1997) sequence differences between nucleotides with high base and their sequences are determined to identify SNPs. PCR is quality value "high-quality mismatches. Results are also performed with primers tagged with 5'-ATT or 5'-GTT at their compared to results of restriction fragment mapping of 60 ends for post-labeling of both strands. DHPLC analysis is genomic clones at Washington University Genome Sequenc carried out using the WAVE DNA fragment analysis system ing Center, finisher's reports on overlaps, and results of the (Transgenomic). PCR products are injected into DNASep sequence contig building effort at the NCBI. SNP detection: column, and separated under the conditions determined using Overlapping pairs of clone sequence are analyzed for candi WAVEMaker program (Transgenomic). The two strands of date SNP Sites with the POLYBAYES SNP detection Soft 65 PCR products that are differentially labeled with nucleotides ware. Sequence differences between the pair of sequences are modified with R110 and R6G by an exchange reaction of scored for the probability of representing true sequence varia Klenow fragment of DNA polymerase I. The reaction is US 8,080,578 B2 39 40 stopped by adding EDTA, and unincorporated nucleotides are amino acid analysis (high pressure liquid chromatography or dephosphorylated by adding calf intestinal alkaline phos mass spectroscopy) could be used that would detect whether phatase. SSCP followed by electrophoresis is performed in a the protein has Arg or Gly. capillary using an ABI Prism 310 Genetic Analyzer. Genes Therefore, in certain embodiments, the present invention can softwares (P-E Biosystems). DNA of individuals includ concerns compositions comprising at least one proteinaceous ing those who showed different genotypes on DHPLC or molecule. Such as a BAR protein or an protein that binds SSCP are subjected for direct sequencing using big-dye ter fAR protein, such as an antibody. As used herein, a “pro minator chemistry, on ABI Prism 310 sequencer. Multiple teinaceous molecule.” “proteinaceous composition.” “pro sequence trace files obtained from ABI Prism 310 are pro teinaceous compound,” “proteinaceous chain' or “proteina 10 ceous material' generally refers, but is not limited to, a cessed and aligned by Phred/Phrap and viewed using Consed protein of greater than about 200 amino acids or the full viewer. SNPs are identified by PolyPhred software and visual length endogenous sequence translated from a gene; a inspection. Trace chromatogram data of EST sequences in polypeptide of greater than about 100 amino acids; and/or a Unigene are processed with PHRED. To identify likely SNPs, peptide of from about 3 to about 100 amino acids. All the single base mismatches are reported from multiple sequence 15 “proteinaceous' terms described above may be used inter alignments produced by the programs PHRAP, BRO and changeably herein. POA for each Unigene cluster. BRO corrected possible mis Proteinaceous compositions may be made by any tech reported EST orientations, while POA identified and ana nique known to those of skill in the art, including the expres lyzed non-linear alignment structures indicative of gene mix sion of proteins, polypeptides or peptides through standard ing/chimeras that might produce spurious SNPs. Bayesian molecular biological techniques, the isolation of proteina inference is used to weigh evidence for true polymorphism ceous compounds from natural sources, or the chemical Syn Versus sequencing error, misalignment or ambiguity, misclus thesis of proteinaceous materials. The nucleotide and protein, tering or chimeric EST sequences, assessing data Such as raw polypeptide and peptide sequences for various genes have chromatogram height, sharpness, overlap and spacing; been previously disclosed, and may be found at computerized sequencing error rates; context-sensitivity; cDNA library ori 25 databases known to those of ordinary skill in the art. One such gin, etc. database is the National Center for Biotechnology Informa In method identified as MARSHFIELD(Method-B), over tions Genbank and GenPept databases (http://www.ncbi.n- lapping human DNA sequences which contained putative lm.nih.gov/). The coding regions for these known genes may insertion/deletion polymorphisms are identified through be amplified and/or expressed using the techniques disclosed searches of public databases. PCR primers which flanked 30 herein or as would be know to those of ordinary skill in the art. each polymorphic site are selected from the consensus Alternatively, various commercial preparations of proteins, sequences. Primers are used to amplify individual or pooled polypeptides and peptides are known to those of skill in the human genomic DNA. Resulting PCR products are resolved art. on a denaturing polyacrylamide gel and a Phosphorlmager is 1. Protein Purification 35 It may be desirable to purify BAR from a sample or purify used to estimate allele frequencies from DNA pools. a protein that binds BAR, Such as an antibody. Such tech f. Linkage Disequilibrium niques are widely employed and the invention is not intended Polymorphisms in linkage disequilibrium with another to be limited with respect to protein purification. Protein polymorphism in which identification of one polymorphism purification techniques are well known to those of skill in the is predictive of the identity of the linked polymorphism. 40 art. These techniques involve, at one level, the crudefraction “Linkage disequilibrium” (“LD’ as used herein, though also ation of the cellular milieu to polypeptide and non-polypep referred to as “LED in the art) refers to a situation where a tide fractions. Having separated the polypeptide from other particular combination of alleles (i.e., a variant form of a proteins, the polypeptide of interest may be further purified given gene) or polymorphisms at two loci appears more fre using chromatographic and electrophoretic techniques to quently than would be expected by chance. “Significant’ as 45 achieve partial or complete purification (or purification to used in respect to linkage disequilibrium, as determined by homogeneity). Analytical methods particularly Suited to the one of skill in the art, is contemplated to be a statistical p or a preparation of a pure peptide are ion-exchange chromatogra value that may be 0.25 or 0.1 and may be 0.1, 0.05. 0.001, phy, exclusion chromatography; polyacrylamide gel electro 0.00001 or less. The polymorphism at position 389 in the phoresis; isoelectric focusing. A particularly efficient method BAR protein may be determined by evaluating the nucleic 50 of purifying peptides is fast protein liquid chromatography or acid sequence of a polymorphism in linkage disequilibrium even HPLC. with the 389 polymorphism. The invention may be imple Certain aspects of the present invention may concern the mented in this manner with respect to one or more polymor purification, and in particular embodiments, the Substantial phisms so as to allow haplotype analysis. “Haplotype' is used purification, of an encoded protein or peptide. The term “puri according to its plain and ordinary meaning to one skilled in 55 fied protein or peptide' as used herein, is intended to refer to the art. It refers to a collective genotype of two or more alleles a composition, isolatable from other components, wherein or polymorphisms along one of the homologous chromo the protein or peptide is purified to any degree relative to its SOS. naturally-obtainable state. A purified protein or peptide there B. Evaluating the Protein fore also refers to a protein or peptide, free from the environ Alternatively, polymorphic variation can be determined by 60 ment in which it may naturally occur. any method that detects an amino acid variation at position Generally, “purified’ will refer to a protein or peptide com 389 of the BAR protein. The invention should not be limited position that has been Subjected to fractionation to remove by any particular method for achieving this. For example, a various other components, and which composition Substan sample of fluid or tissue may be obtained from an individual tially retains its expressed biological activity. Where the term and the amino acid at position 389 of the BAR protein is 65 “substantially purified' is used, this designation will refer to determined. Such detection can be by various methods a composition in which the protein or peptide forms the major including antibody based assays, (Western blots, ELISA) or component of the composition, such as constituting about US 8,080,578 B2 41 42 50%, about 60%, about 70%, about 80%, about 90%, about which the particles are made does not adsorb the molecules, 95% or more of the proteins in the composition. the sole factor determining rate of flow is the size. Hence, Various methods for quantifying the degree of purification molecules are eluted from the column in decreasing size, so of the protein or peptide will be known to those of skill in the long as the shape is relatively constant. Gel chromatography art in light of the present disclosure. These include, for is unsurpassed for separating molecules of different size example, determining the specific activity of an active frac because separation is independent of all other factors such as tion, or assessing the amount of polypeptides within a fraction pH, ionic strength, temperature, etc. There also is virtually no by SDS/PAGE analysis. A preferred method for assessing the adsorption, less Zone spreading and the elution Volume is purity of a fraction is to calculate the specific activity of the related in a simple matter to molecular weight. fraction, to compare it to the specific activity of the initial 10 Affinity Chromatography is a chromatographic procedure extract, and to thus calculate the degree of purity, herein that relies on the specific affinity between a substance to be assessed by a “-fold purification number.” The actual units isolated and a molecule that it can specifically bind to. This is used to represent the amount of activity will, of course, be a receptor-ligand type interaction. The column material is dependent upon the particular assay technique chosen to fol synthesized by covalently coupling one of the binding part low the purification and whether or not the expressed protein 15 ners to an insoluble matrix. The column material is then able or peptide exhibits a detectable activity. to specifically adsorb the substance from the solution. Elution A variety of techniques suitable for use in protein purifi occurs by changing the conditions to those in which binding cation will be well known to those of skill in the art. These will not occur (e.g., alter pH, ionic strength, and temperature). include, for example, precipitation with ammonium Sulfate, A particular type of affinity chromatography useful in the PEG, antibodies and the like or by heat denaturation, fol purification of carbohydrate containing compounds is lectin lowed by centrifugation; chromatography steps such as ion affinity chromatography. Lectins area class of substances that exchange, gel filtration, reverse phase, hydroxylapatite and bind to a variety of polysaccharides and glycoproteins. Lec affinity chromatography; isoelectric focusing; gel electro tins are usually coupled to agarose by cyanogen bromide. phoresis; and combinations of such and other techniques. As Conconavalin A coupled to Sepharose was the first material is generally known in the art, it is believed that the order of 25 of this sort to be used and has been widely used in the isolation conducting the various purification steps may be changed, or of polysaccharides and glycoproteins other lectins that have that certain steps may be omitted, and still result in a Suitable been include lentil lectin, wheat germ agglutinin which has method for the preparation of a substantially purified protein been useful in the purification of N-acetylglucosaminyl resi or peptide. dues and Helix pomatia lectin. Lectins themselves are puri There is no general requirement that the protein or peptide 30 fied using affinity chromatography with carbohydrate always be provided in their most purified state. Indeed, it is ligands. Lactose has been used to purify lectins from castor contemplated that less substantially purified products will bean and peanuts; maltose has been useful in extracting lec have utility in certain embodiments. Partial purification may tins from lentils and jack bean: N-acetyl-D-galactosamine is be accomplished by using fewer purification steps in combi used for purifying lectins from Soybean: N-acetylglucosami nation, or by utilizing different forms of the same general 35 nyl binds to lectins from wheat germ; D-galactosamine has purification scheme. For example, it is appreciated that a been used in obtaining lectins from clams and L-fucose will cation-exchange column chromatography performed utiliz bind to lectins from lotus. ing an HPLC apparatus will generally result in a greater The matrix should be a substance that itself does not adsorb “-fold' purification than the same technique utilizing a low molecules to any significant extent and that has a broad range pressure chromatography system. Methods exhibiting a 40 of chemical, physical and thermal stability. The ligand should lower degree of relative purification may have advantages in be coupled in Such a way as to not affect its binding proper total recovery of protein product, or in maintaining the activ ties. The ligand also should provide relatively tight binding. ity of an expressed protein. And it should be possible to elute the substance without It is known that the migration of a polypeptide can vary, destroying the sample or the ligand. One of the most common sometimes significantly, with different conditions of SDS/ 45 forms of affinity chromatography is immunoaffinity chroma PAGE (Capaldi et al., 1977). It will therefore be appreciated tography. The generation of antibodies that would be suitable that under differing electrophoresis conditions, the apparent for use in accord with the present invention is discussed molecular weights of purified or partially purified expression below. products may vary. 2. Antibodies High Performance Liquid Chromatography (HPLC) is 50 Another embodiment of the present invention are antibod characterized by a very rapid separation with extraordinary ies, in Some cases, a human monoclonal antibody immunore resolution of peaks. This is achieved by the use of very fine active with the polypeptide sequence of human BAR. It is particles and high pressure to maintain an adequate flow rate. understood that antibodies can be used for detecting fBAR, Separation can be accomplished in a matter of minutes, or at particularly a? AR that is the result of a particular polymor most an hour. Moreover, only a very small volume of the 55 phism. It is contemplated that antibodies particularly useful in sample is needed because the particles are so Small and close the context of the present invention are those that differen packed that the void volume is a very small fraction of the bed tially binda BAR protein with a Gly389 compared to a BAR Volume. Also, the concentration of the sample need not be protein with a Arg,389 so as to distinguish between the two very great because the bands are so narrow that there is very populations. little dilution of the sample. 60 As used herein, the term “antibody' is intended to refer Gel chromatography, or molecular sieve chromatography, broadly to any immunologic binding agent such as IgG, IgM, is a special type of partition chromatography that is based on IgA, Ig|D and IgE. Generally, IgG and/or IgM are preferred molecular size. The theory behind gel chromatography is that because they are the most common antibodies in the physi the column, which is prepared with tiny particles of an inert ological situation and because they are most easily made in a Substance that contain Small pores, separates larger molecules 65 laboratory setting. from Smaller molecules as they pass through or around the The term “antibody' is used to refer to any antibody-like pores, depending on their size. As long as the material of molecule that has an antigen binding region, and includes US 8,080,578 B2 43 44 antibody fragments such as Fab', Fab., F(ab'), single domain (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). antibodies (DABs). Fv, scfv (single chain Fv), and the like. RIBI, which contains three components extracted from bac The techniques for preparing and using various antibody teria, MPL, trehalose dimycolate (TDM) and cell wall skel based constructs and fragments are well known in the art. eton (CWS) in a 2% squalene/Tween 80 emulsion also is Means for preparing and characterizing antibodies are also contemplated. MHC antigens may even be used. Exemplary, well known in the art (See, e.g., Harlow et al., 1988; incor often preferred adjuvants include complete Freund's adju porated herein by reference). vant (a non-specific stimulator of the immune response con a. Antibody Generation taining killed Mycobacterium tuberculosis), incomplete Fre In certain embodiments, the present invention involves unds adjuvants and aluminum hydroxide adjuvant. antibodies. For example, all or part of a monoclonal may be 10 In addition to adjuvants, it may be desirable to coadminis used in determining the amino acid at position 389. As ter biologic response modifiers (BRM), which have been detailed above, in addition to antibodies generated against shown to upregulate T cell immunity or down-regulate Sup full length proteins, antibodies also may be generated in pressor cell activity. Such BRMs include, but are not limited response to Smaller constructs comprising epitopic core to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.); low regions, including wild-type and mutant epitopes. The tech 15 dose Cyclophosphamide (CYP300 mg/m) (Johnson/Mead, niques for preparing and using various antibody-based con N.J.), cytokines Such as Y-interferon, IL-2, or IL-12 or genes structs and fragments are well known in the art. Means for encoding proteins involved in immune helper functions. Such preparing and characterizing antibodies are also well known as B-7. in the art (See, e.g., Harlow and Lane, 1988; incorporated The amount of immunogen composition used in the pro herein by reference). duction of polyclonal antibodies varies upon the nature of the Monoclonal antibodies (mAbs) are recognized to have cer immunogen as well as the animal used for immunization. A tain advantages, e.g., reproducibility and large-scale produc variety of routes can be used to administer the immunogen tion, and their use is generally preferred. The invention thus (Subcutaneous, intramuscular, intradermal, intravenous and provides monoclonal antibodies of the human, murine, mon intraperitoneal). The production of polyclonal antibodies key, rat, hamster, rabbit and even chicken origin. 25 may be monitored by sampling blood of the immunized ani The methods for generating monoclonal antibodies mal at various points following immunization. (mAbs) generally begin along the same lines as those for A second, booster injection also may be given. The process preparing polyclonal antibodies. Briefly, a polyclonal anti of boosting and titering is repeated until a suitable titer is body may be prepared by immunizing an animal with an achieved. When a desired level of immunogenicity is immunogenic polypeptide composition in accordance with 30 obtained, the immunized animal can be bled and the serum the present invention and collecting antisera from that immu isolated and stored, and/or the animal can be used to generate nized animal. Alternatively, in some embodiments of the mAbs. present invention, serum is collected from persons who may mAbs may be readily prepared through use of well-known have been exposed to a particular antigen. Exposure to a techniques, such as those exemplified in U.S. Pat. No. 4,196, particular antigen may occur a work environment, such that 35 265, incorporated herein by reference. Typically, this tech those persons have been occupationally exposed to a particu nique involves immunizing a suitable animal with a selected lar antigen and have developed polyclonal antibodies to a immunogen composition, e.g., a purified or partially purified peptide, polypeptide, or protein. In some embodiments of the polypeptide, peptide or domain, be it a wild-type or mutant invention polyclonal serum from occupationally exposed per composition. The immunizing composition is administered Sons is used to identify antigenic regions in the gelonin toxin 40 in a manner effective to stimulate antibody producing cells. through the use of immunodetection methods. mAbs may be further purified, if desired, using filtration, A wide range of animal species can be used for the pro centrifugation and various chromatographic methods such as duction of antisera. Typically the animal used for production HPLC or affinity chromatography. Fragments of the mono of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig clonal antibodies of the invention can be obtained from the or a goat. Because of the relatively large blood volume of 45 monoclonal antibodies so produced by methods which rabbits, a rabbit is a preferred choice for production of poly include digestion with enzymes, such as pepsin or papain, clonal antibodies. and/or by cleavage of disulfide bonds by chemical reduction. As is well known in the art, a given composition may vary Alternatively, monoclonal antibody fragments encompassed in its immunogenicity. It is often necessary therefore to boost by the present invention can be synthesized using an auto the host immune system, as may be achieved by coupling a 50 mated peptide synthesizer. peptide or polypeptide immunogen to a carrier. Exemplary It also is contemplated that a molecular cloning approach and preferred carriers are keyhole limpet hemocyanin (KLH) may be used to generate mAbs. For this, combinatorial immu and bovine serum albumin (BSA). Other albumins such as noglobulin phagemid libraries are prepared from RNA iso ovalbumin, mouse serum albumin or rabbit serum albumin lated from the spleen of the immunized animal, and also can be used as carriers. Means for conjugating a polypep 55 phagemids expressing appropriate antibodies are selected by tide to a carrier protein are well known in the art and include panning using cells expressing the antigen and control cells. glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinim The advantages of this approach over conventional hybri ide ester, carbodiimide and bis-biazotized benzidine. doma techniques are that approximately 10 times as many As also well known in the art, the immunogenicity of a antibodies can be produced and Screened in a single round, particular immunogen composition can be enhanced by the 60 and that new specificities are generated by H and L chain use of non-specific stimulators of the immune response, combination which further increases the chance of finding known as adjuvants. Suitable molecule adjuvants include all appropriate antibodies. acceptable immunostimulatory compounds, such as cytok b. Immunodetection Methods ines, toxins or synthetic compositions. As discussed, in some embodiments, the present invention Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, 65 concerns immunodetection methods for binding, purifying, IL-12, Y-interferon, GMCSP, BCG, aluminum hydroxide, removing, determining, and/or otherwise detecting biologi MDP compounds, such as thur-MDP and nor-MDP, CGP cal components such as antigenic regions on polypeptides US 8,080,578 B2 45 46 and peptides. The immunodetection methods of the present Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996.345; 4,277, invention can be used to identify antigenic regions of a pep 437; 4,275,149 and 4.366,241, each incorporated herein by tide, polypeptide, or protein that has therapeutic implications, reference. Of course, one may find additional advantages particularly in reducing the immunogenicity or antigenicity through the use of a secondary binding ligand Such as a of the peptide, polypeptide, or protein in a target Subject. 5 second antibody and/or a biotin/avidin ligand binding Immunodetection methods include enzyme linked immu arrangement, as is known in the art. nosorbent assay (ELISA), radioimmunoassay (RIA), immu The antibody employed in the detection may itself be noradiometric assay, fluoroimmunoassay, chemiluminescent linked to a detectable label, wherein one would then simply assay, bioluminescent assay, and Western blot, though several detect this label, thereby allowing the amount of the primary others are well known to those of ordinary skill. The steps of 10 immune complexes in the composition to be determined. various useful immunodetection methods have been Alternatively, the first antibody that becomes bound within described in the scientific literature, such as, e.g., Doolittle et the primary immune complexes may be detected by means of al., 1999; Gulbis et al., 1993: De Jager et al., 1993; and a second binding ligand that has binding affinity for the anti Nakamura et al., 1987, each incorporated herein by reference. body. In these cases, the second binding ligand may be linked In general, the immunobinding methods include obtaining 15 to a detectable label. The second binding ligand is itself often a sample Suspected of containing a protein, polypeptide and/ an antibody, which may thus be termed a “secondary anti or peptide, and contacting the sample with a first antibody, body. The primary immune complexes are contacted with the monoclonal or polyclonal, in accordance with the present labeled, secondary binding ligand, or antibody, under effec invention, as the case may be, under conditions effective to tive conditions and for a period of time sufficient to allow the allow the formation of immunocomplexes. formation of secondary immune complexes. The secondary These methods include methods for purifying a protein, immune complexes are then generally washed to remove any polypeptide and/or peptide from organelle, cell, tissue or non-specifically bound labeled secondary antibodies or organism's samples. In these instances, the antibody removes ligands, and the remaining label in the secondary immune the antigenic protein, polypeptide and/or peptide component complexes is then detected. from a sample. The antibody will preferably be linked to a 25 Further methods include the detection of primary immune Solid Support. Such as in the form of a column matrix, and the complexes by a two step approach. A second binding ligand, sample Suspected of containing the protein, polypeptide and/ Such as an antibody, that has binding affinity for the antibody or peptide antigenic component will be applied to the immo is used to form secondary immune complexes, as described bilized antibody. The unwanted components will be washed above. After washing, the secondary immune complexes are from the column, leaving the antigen immunocomplexed to 30 contacted with a third binding ligand or antibody that has the immobilized antibody to be eluted. binding affinity for the second antibody, again under effective The immunobinding methods also include methods for conditions and for a period of time sufficient to allow the detecting and quantifying the amount of an antigen compo formation of immune complexes (tertiary immune com nent in a sample and the detection and quantification of any plexes). The third ligand or antibody is linked to a detectable immune complexes formed during the binding process. Here, 35 label, allowing detection of the tertiary immune complexes one would obtain a sample Suspected of containing an antigen thus formed. This system may provide for signal amplifica orantigenic domain, and contact the sample with an antibody tion if this is desired. against the antigen or antigenic domain, and then detect and One method of immunodetection designed by Charles quantify the amount of immune complexes formed under the Cantor uses two different antibodies. A first step biotinylated, specific conditions. 40 monoclonal or polyclonal antibody is used to detect the target In terms of antigen detection, the biological sample ana antigen?s), and a second step antibody is then used to detect lyzed may be any sample that is suspected of containing an the biotin attached to the complexed biotin. In that method the antigen or antigenic domain, such as, for example, a tissue sample to be tested is first incubated in a solution containing section or specimen, a homogenized tissue extract, a cell, an the first step antibody. If the target antigen is present, Some of organelle, separated and/or purified forms of any of the above 45 the antibody binds to the antigen to form a biotinylated anti antigen-containing compositions, or even any biological fluid body/antigen complex. The antibody/antigen complex is then that comes into contact with the cellor tissue, including blood amplified by incubation in Successive Solutions of Streptavi and/or serum. din (or avidin), biotinylated DNA, and/or complementary Contacting the chosen biological sample with the antibody biotinylated DNA, with each step adding additional biotin under effective conditions and for a period of time sufficient 50 sites to the antibody/antigen complex. The amplification to allow the formation of immune complexes (primary steps are repeated until a suitable level of amplification is immune complexes) is generally a matter of simply adding achieved, at which point the sample is incubated in a solution the antibody composition to the sample and incubating the containing the second step antibody against biotin. This sec mixture for a period of time long enough for the antibodies to ond step antibody is labeled, as for example with an enzyme form immune complexes with, i.e., to bind to, any antigens 55 that can be used to detect the presence of the antibody/antigen present. After this time, the sample-antibody composition, complex by histoenzymology using a chromogen Substrate. such as a tissue section, ELISA plate, dot blot or western blot, With Suitable amplification, a conjugate can be produced will generally be washed to remove any non-specifically which is macroscopically visible. bound antibody species, allowing only those antibodies spe Another known method of immunodetection takes advan cifically bound within the primary immune complexes to be 60 tage of the immuno-PCR (Polymerase Chain Reaction) meth detected. odology. The PCR method is similar to the Cantor method up In general, the detection of immunocomplex formation is to the incubation with biotinylated DNA, however, instead of well known in the art and may be achieved through the appli using multiple rounds of streptavidin and biotinylated DNA cation of numerous approaches. These methods are generally incubation, the DNA/biotin/streptavidin/antibody complex is based upon the detection of a label or marker, such as any of 65 washed out with a low pH or high saltbuffer that releases the those radioactive, fluorescent, biological and enzymatic tags. antibody. The resulting wash solution is then used to carry out U.S. patents concerning the use of such labels include U.S. a PCR reaction with suitable primers with appropriate con US 8,080,578 B2 47 48 trols. At least in theory, the enormous amplification capability and thus reduces the background caused by nonspecific bind and specificity of PCR can be utilized to detect a single ing of antisera onto the Surface. antigen molecule. In ELISAS, it is probably more customary to use a second i. ELISAS ary or tertiary detection means rather than a direct procedure. As detailed above, immunoassays, in their most simple Thus, after binding of a protein or antibody to the well, and/or direct sense, are binding assays. Certain preferred coating with a non-reactive material to reduce background, immunoassays are the various types of enzyme linked immu and washing to remove unbound material, the immobilizing nosorbent assays (ELISAS) and/or radioimmunoassays surface is contacted with the biological sample to be tested (RIA) known in the art. Immunohistochemical detection under conditions effective to allow immune complex (anti using tissue sections is also particularly useful. However, it 10 gen/antibody) formation. Detection of the immune complex will be readily appreciated that detection is not limited to such then requires a labeled secondary binding ligand orantibody, techniques, and/or western blotting, dot blotting, FACS and a secondary binding ligand or antibody in conjunction analyses, and/or the like may also be used. with a labeled tertiary antibody or a third binding ligand. In one exemplary ELISA, antibodies are immobilized onto “Under conditions effective to allow immune complex (an a selected Surface exhibiting protein affinity, such as a well in 15 tigen/antibody) formation' means that the conditions prefer a polystyrene microtiter plate. Then, a test composition Sus ably include diluting the antigens and/orantibodies with solu pected of containing the antigen, Such as a clinical sample, is tions such as BSA, bovine gamma globulin (BGG) or added to the wells. After binding and/or washing to remove phosphate buffered saline (PBS)/Tween. These added agents non-specifically bound immune complexes, the bound anti also tend to assist in the reduction of nonspecific background. gen may be detected. Detection is generally achieved by the The "suitable' conditions also mean that the incubation is addition of another antibody that is linked to a detectable at a temperature or for a period of time sufficient to allow label. This type of ELISA is a simple “sandwich ELISA.” effective binding. Incubation steps are typically from about 1 Detection may also be achieved by the addition of a second to 2 to 4 hours or so, at temperatures preferably on the order antibody, followed by the addition of a third antibody that has of 25°C. to 27°C., or may be overnight at about 4°C. or so. binding affinity for the second antibody, with the third anti 25 Following all incubation steps in an ELISA, the contacted body being linked to a detectable label. The ELISA may be Surface is washed so as to remove non-complexed material. based on differential binding of an antibody to a protein with An example of a washing procedure includes washing with a Arg,389 versus Gly389. solution such as PBS/Tween, or borate buffer. Following the In another exemplary ELISA, the samples suspected of formation of specific immune complexes between the test containing the antigen are immobilized onto the well Surface 30 sample and the originally bound material, and Subsequent and/or then contacted with antibodies. After binding and/or washing, the occurrence of even minute amounts of immune washing to remove non-specifically bound immune com complexes may be determined. plexes, the bound anti-antibodies are detected. Where the To provide a detecting means, the second or third antibody initial antibodies are linked to a detectable label, the immune will have an associated label to allow detection. This may be complexes may be detected directly. Again, the immune com 35 an enzyme that will generate color development upon incu plexes may be detected using a second antibody that has bating with an appropriate chromogenic Substrate. Thus, for binding affinity for the first antibody, with the second anti example, one will desire to contact or incubate the first and body being linked to a detectable label. second immune complex with a urease, glucose oxidase, Another ELISA in which the antigens are immobilized, alkaline phosphatase or hydrogen peroxidase-conjugated involves the use of antibody competition in the detection. In 40 antibody for a period of time and under conditions that favor this ELISA, labeled antibodies againstan antigen are added to the development of further immune complex formation (e.g., the wells, allowed to bind, and/or detected by means of their incubation for 2 hours at room temperature in a PBS-contain label. The amount of an antigen in an unknown sample is then ing solution such as PBS-Tween). determined by mixing the sample with the labeled antibodies After incubation with the labeled antibody, and subsequent against the antigen during incubation with coated wells. The 45 to washing to remove unbound material, the amount of label presence of an antigen in the sample acts to reduce the amount is quantified, e.g., by incubation with a chromogenic Sub of antibody against the antigen available for binding to the strate such as urea, or bromocresol purple, or 2,2'-azino-di well and thus reduces the ultimate signal. This is also appro (3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or HO, in priate for detecting antibodies against an antigen in an the case of peroxidase as the enzyme label. Quantification is unknown sample, where the unlabeled antibodies bind to the 50 then achieved by measuring the degree of color generated, antigen-coated wells and also reduces the amount of antigen e.g., using a visible spectra spectrophotometer. available to bind the labeled antibodies. ii. Immunohistochemistry Irrespective of the format employed, ELISAs have certain The antibodies of the present invention may also be used in features in common, Such as coating, incubating and binding, conjunction with both fresh-frozen and/or formalin-fixed, washing to remove non-specifically bound species, and 55 paraffin-embedded tissue blocks prepared for study by immu detecting the bound immune complexes. These are described nohistochemistry (IHC). For example, immunohistochemis below. try may be utilized to characterize Fortilin or to evaluate the In coating a plate with either antigen or antibody, one will amount Fortilin in a cell. The method of preparing tissue generally incubate the wells of the plate with a solution of the blocks from these particulate specimens has been Success antigen orantibody, either overnight or for a specified period 60 fully used in previous IHC studies of various prognostic fac of hours. The wells of the plate will then be washed to remove tors, and/or is well known to those of skill in the art (Brown et incompletely adsorbed material. Any remaining available al., 1990; Abbondanzo et al., 1990; Allred et al., 1990). surfaces of the wells are then “coated with a nonspecific Briefly, frozen-sections may be prepared by rehydrating 50 protein that is antigenically neutral with regard to the test mg of frozen “pulverized’ tissue at room temperature in antisera. These include bovine serum albumin (BSA), casein 65 phosphate buffered saline (PBS) in small plastic capsules: or Solutions of milk powder. The coating allows for blocking pelleting the particles by centrifugation; resuspending them of nonspecific adsorption sites on the immobilizing Surface in a Viscous embedding medium (OCT); inverting the capsule US 8,080,578 B2 49 50 and/or pelleting again by centrifugation: Snap-freezing in binant cells are introduced into a patient. Aqueous composi -70° C. isopentane; cutting the plastic capsule and/or remov tions of the present invention comprise an effective amount of ing the frozen cylinder of tissue; securing the tissue cylinder the vector or cells, dissolved or dispersed in a pharmaceuti on a cryostat microtome chuck; and/or cutting 25-50 serial cally acceptable carrier or aqueous medium. The phrase sections. “pharmaceutically or pharmacologically acceptable' refer to Permanent-sections may be prepared by a similar method molecular entities and compositions that do not produce involving rehydration of the 50 mg sample in a plastic adverse, allergic, or other untoward reactions when adminis microfuge tube; pelleting; resuspending in 10% formalin for tered to an animal or a human. As used herein, pharmaceu 4 hours fixation; washing/pelleting; resuspending in warm tically acceptable carrier includes solvents, buffers, solu 2.5% agar; pelleting; cooling in ice water to harden the agar, 10 tions, dispersion media, coatings, antibacterial and antifungal removing the tissue?agar block from the tube; infiltrating agents, isotonic and absorption delaying agents and the like and/or embedding the block in paraffin; and/or cutting up to acceptable for use in formulating pharmaceuticals, such as 50 serial permanent sections. pharmaceuticals suitable for administration to humans. The III. Therapy use of Such media and agents for pharmaceutically active Once the genotype or the protein sequence off-AR of the 15 Substances is well known in the art. Except insofar as any individual is determined a therapeutic course of treatment conventional media or agent is incompatible with the active may be individualized. In a preferred embodiment of the ingredients of the present invention, its use in therapeutic method, the trait of interest is a clinical response exhibited by compositions is contemplated. Supplementary active ingre a patient to Some therapeutic treatment, for example, dients also can be incorporated into the compositions, pro response to a drug Such as but not limited to a B-blocker, Such vided they do not inactivate the vectors or cells of the com as bucindolol, targeting faR or response to a therapeutic positions. treatment for a medical condition. As used herein, "medical The active compositions of the present invention may condition' includes but is not limited to any condition or include classic pharmaceutical preparations. Administration disease manifested as one or more physical and/or psycho of these compositions according to the present invention may logical symptoms for which treatment is desirable, and 25 be via any common route so long as the target tissue is includes previously and newly identified diseases and other available via that route. This includes oral, nasal, or buccal. disorders having similar pathophysiological states, such as Alternatively, administration may be by intradermal, Subcu but not limited to, heart failure, pheochromocytoma, taneous, intramuscular, intraperitoneal or intravenous injec migraines, cardiac arrhythmias, hypertension, dilated cardi tion, or by direct injection into cardiac tissue. Such compo omyopathy, ischemic heart disease (cardiomyopathy, 30 sitions would normally be administered as pharmaceutically ischemic heart disease (cardiomyopathy, angina, myocardial acceptable compositions, as described Supra. infarction), and various anxiety disorders. As used herein the The active compounds may also be administered parenter term “clinical response’ means any or all of the following: a ally or intraperitoneally. By way of illustration, solutions of quantitative measure of the efficacy or potency of the therapy the active compounds as free base or pharmacologically and adverse events (i.e., side effects). 35 acceptable salts can be prepared in water suitably mixed with Thus homozygous BArg389 individuals having a medical a Surfactant, Such as hydroxypropylcellulose. Dispersions condition can be placed on a therapy that includes B-blockers can also be prepared in glycerol, liquid polyethylene glycols, such as but not limited to bucindolol. The B-blocker may be and mixtures thereof and in oils. Under ordinary conditions of administered alone or in combination with at least one other storage and use, these preparations generally contain a pre agent, such as a stabilizing compound. The B-blocker may 40 servative to prevent the growth of microorganisms. also be administered in combination with a medical device The pharmaceutical forms suitable for injectable use that would have previously been contraindicated by the dis include, for example, Sterile aqueous solutions or dispersions ease state that required the device. For example, normally a and sterile powders for the extemporaneous preparation of heart failure patient with bradycardia would not receive a sterile injectable solutions or dispersions. Generally, these B-blocker. But if the genotype of the individual is Arg389 (the 45 preparations are sterile and fluid to the extent that easy inject favorable genotype) a pacemaker could be implanted, to pre ability exists. Preparations should be stable under the condi scribe bucindolol. tions of manufacture and storage and should be preserved A. Routes of Administration against the contaminating action of microorganisms. Such as Administration of the B-blocker may be by any number of bacteria and fungi. Appropriate solvents or dispersion media routes including, but not limited to oral, intravenous, intra 50 may contain, for example, water, ethanol, polyol (for muscular, intra-arterial, intramedullary, intrathecal, intraven example, glycerol, propylene glycol, and liquid polyethylene tricular, intradermal, intratracheal, intravesicle, intraocular, glycol, and the like), Suitable mixtures thereof, and vegetable transdermal. Subcutaneous, intraperitoneal, intranasal, oils. The proper fluidity can be maintained, for example, by enteral, topical, Sublingual, or rectal. Further details on tech the use of a coating, such as lecithin, by the maintenance of niques for formulation and administration may be found in 55 the required particle size in the case of dispersion and by the the latest edition of Remington’s Pharmaceutical Sciences use of surfactants. The prevention of the action of microor (Maack Publishing Co., Easton, Pa.). In certain embodiments ganisms can be brought about by various antibacterial an bucindolol is formulated for oral administration. antifungal agents, for example, parabens, chlorobutanol, phe B. Formulations nol, Sorbic acid, thimerosal, and the like. In many cases, it will Where clinical applications are contemplated, pharmaceu 60 be preferable to include isotonic agents, for example, Sugars tical compositions will be prepared in a form appropriate for or sodium chloride. Prolonged absorption of the injectable the intended application. Generally, this will entail preparing compositions can be brought about by the use in the compo compositions that are essentially free of pyrogens, as well as sitions of agents delaying absorption, for example, aluminum other impurities that could be harmful to humans or animals. monostearate and gelatin. One will generally desire to employ appropriate salts and 65 For oral administration the polypeptides of the present buffers to render delivery vectors stable and allow for uptake invention generally may be incorporated with excipients and by target cells. Buffers also will be employed when recom used in the form of non-ingestible mouthwashes and denti US 8,080,578 B2 51 52 frices. A mouthwash may be prepared incorporating the tical binder, about 2-20% of a hydrophobic component such active ingredient in the required amount in an appropriate as a waxy material, e.g., a fatty acid, and about 30-90% active solvent, such as a sodium borate solution (Dobell's Solution). ingredient. Alternatively, the active ingredient may be incorporated into b. Films an antiseptic wash containing sodium borate, glycerin and 5 This invention further provides a prophylaxis for or potassium bicarbonate. The active ingredient may also be method of treating a patient having a homozygous B1Arg389 dispersed in dentifrices, including: gels, pastes, powders and genotype following an invasive cardiac procedure comprising slurries. The active ingredient may be added in a therapeuti administering biodegradable, biocompatible polymeric film cally effective amount to a paste dentifrice that may include comprising a 3-blocker, Such as bucindolol, to a patient. The water, binders, abrasives, flavoring agents, foaming agents, 10 polymeric films are thin compared to their length and breadth. and humectants. The films typically have a uniform selected thickness between about 60 micrometers and about 5 mm. Films of The compositions of the present invention generally may between about 600 micrometers and 1 mm and between about be formulated in a neutral or salt form. Pharmaceutically 1 mmandabout 5 mm thick, as well as films between about 60 acceptable salts include, for example, acid addition salts 15 micrometers and about 1000 micrometers, and between about (formed with the free amino groups of the protein) derived 60 and about 300 micrometers are useful in the manufacture from inorganic acids (e.g., hydrochloric or phosphoric acids, of therapeutic implants for insertion into a patient’s body. The or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, films can be administered to the patient in a manner similar to and the like. Salts formed with the free carboxyl groups of the methods used in adhesion Surgeries. For example, a protein can also be derived from inorganic bases (e.g., B-blocker, such as bucindolol, film formulation can be Sodium, potassium, ammonium, calcium, or ferric hydrox sprayed or dropped onto a cardiac tissue site or artery during ides) or from organic bases (e.g., isopropylamine, trimethy Surgery, or a formed film can be placed over the selected tissue lamine, histidine, procaine and the like. site. In an alternative embodiment, the film can be used as Upon formulation, solutions are preferably administered in controlled release coating on a medical device Such as a stent, a manner compatible with the dosage formulation and in Such 25 as is discussed in further detail below. amount as is therapeutically effective. The formulations may Either biodegradable or nonbiodegradable polymers may easily be administered in a variety of dosage forms such as be used to fabricate implants in which the B-blocker is uni injectable solutions, drug release capsules and the like. For formly distributed throughout the polymer matrix. A number parenteral administration in an aqueous solution, for of Suitable biodegradable polymers for use in making the 30 biodegradable films of this invention are known to the art, example, the solution generally is suitably buffered and the including polyanhydrides and aliphatic polyesters, preferably liquid diluent first rendered isotonic for example with suffi polylactic acid (PLA), polyglycolic acid (PGA) and mixtures cient saline or glucose. Such aqueous solutions may be used, and copolymers thereof, more preferably 50:50 copolymers for example, for intravenous, intramuscular, Subcutaneous of PLA:PGA and most preferably 75:25 copolymers of PLA: and intraperitoneal administration. Preferably, Sterile aque 35 PGA. Single enantiomers of PLA may also be used, prefer ous media are employed as is known to those of skill in the art, ably L-PLA, either alone or in combination with PGA. Poly particularly in light of the present disclosure. By way of carbonates, polyfumarates and caprolactones may also be illustration, a single dose may be dissolved in 1 ml of isotonic used to make the implants of this invention. NaCl solution and either added to 1000 ml of hypodermocly The amount of the B-blocker, such as bucindolol, to be sis fluid or injected at the proposed site of infusion, (see for 40 incorporated into the polymeric films of this invention is an example, “Remington’s Pharmaceutical Sciences' 15th Edi amount effective to show a measurable effect in treating dis tion, pages 1035-1038 and 1570-1580). Some variation in eases having similar pathophysiological states, such as but dosage will necessarily occur depending on the condition of not limited to, heart failure, pheochromocytoma, migraines, the subject being treated. The person responsible for admin cardiac arrhythmias, hypertension, aschemia, cardiomyopa istration will, in any event, determine the appropriate dose for 45 thy, and various anxiety disorders. The composition of the the individual subject. Moreover, for human administration, present invention can be incorporated into the film by various preparations should meet Sterility, pyrogenicity, general techniques such as by solution methods, Suspension methods, safety and purity standards as required by FDA Office of or melt pressing. Biologics standards. c. Transdermal Patch Device 1. Controlled/Extended/Sustained/Prolonged Release 50 Transdermal delivery involves delivery of a therapeutic Administration agent through the skin for distribution within the body by Another aspect of this invention provides methods of treat circulation of the blood. Transdermal delivery can be com ing heart failure patients by delivering the B-blocker to a pared to continuous, controlled intravenous delivery of a drug patient, having a homozygous B1Arg389 genotype, as a con using the skin as a port of entry instead of an intravenous trolled release formulation. As used herein, the terms “con 55 needle. The therapeutic agent passes through the outer layers trolled.” “extended,” “sustained, or “prolonged’ release of of the skin, diffuses into the capillaries or tiny blood vessels in the composition of the present invention will collectively be the skin and then is transported into the main circulatory referred to herein as “controlled release, and includes con system. tinuous or discontinuous, and linear or non-linear release of Transdermal patch devices which provide a controlled, the composition of the present invention. There are many 60 continuous administration of a therapeutic agent through the advantages for a controlled release formulation of B-blockers. skin are well known in the art. Such devices, for example, are a. Tablets disclosed in U.S. Pat. Nos. 4,627,429; 4,784.857: 5,662,925; A controlled release tablet suitable for purposes of this 5,788,983; and 6,113,940, which are all incorporated herein invention is disclosed in U.S. Pat. No. 5,126,145, which is by reference. Characteristically, these devices contain a drug incorporated by reference herein. This tablet comprises, in 65 impermeable backing layer which defines the outer surface of admixture, about 5-30% high viscosity hydroxypropyl the device and a permeable skin attaching membrane, such as methyl cellulose, about 2-15% of a water-soluble pharmaceu an adhesive layer, sealed to the barrier layer in Such a way as US 8,080,578 B2 53 54 to create a reservoir between them in which the therapeutic weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or agent is placed. In one embodiment of the present invention a any range derivable therein. Alternatively, it may be admin formulation of the B-blocker is introduced into the reservoir istered systemically over any such period of time and be of a transdermal patch and used by a patient who is homozy extended beyond more than a year. gous Arg389 at the B1AR genes. D. Other Therapeutic Options d. Medical Devices In certain embodiments of the invention, methods may Another embodiment contemplates the incorporation of a involve administering a beta blocker that is not bucindolol or B-blocker, such as bucindolol, into a medical device that is that is an ionotrope, a diuretic, ACE-I, All antagonist, BNP. then positioned to a desired target location within the body, Ca"-blocker, or an HDAC inhibitor. These agents may be whereupon the B-blocker elutes from the medical device. As 10 used herein, "medical device' refers to a device that is intro prescribed or administered instead of or in addition to bucin duced temporarily or permanently into a mammal for the dolol after the f-AR and/or C-AR polymorphisms are prophylaxis or therapy of a medical condition. These devices evaluated. include any that are introduced Subcutaneously, percutane As a second therapeutic regimen, the agent may be admin ously or Surgically to rest within an organ, tissue or lumen. 15 istered or taken at the same time as bucindolol, or either Medical devices include, but are not limited to, stents, syn before or after bucindolol. The treatment may improve one or thetic grafts, artificial heart valves, artificial hearts and fix more symptoms of pathologic cardiac hypertrophy or heart tures to connect the prosthetic organ to the vascular circula failure Such as providing increased exercise capacity, tion, Venous valves, abdominal aortic aneurysm (AAA) increased cardiac ejection Volume, decreased left ventricular grafts, inferior Venal caval filters, catheters including perma end diastolic pressure, decreased pulmonary capillary wedge nent drug infusion catheters, embolic coils, embolic materials pressure, increased cardiac output or cardiac index, lowered used in vascular embolization (e.g., PVA foams), mesh repair pulmonary artery pressures, decreased left ventricular end materials, a Dracon Vascular particle orthopedic metallic systolic and diastolic dimensions, decreased left and right plates, rods and screws and vascular Sutures. ventricular wall stress, decreased wall tension and wall thick In one embodiment, the medical device such as a stent or 25 ness, increased quality of life, and decreased disease-related graft is coated with a matrix. The matrix used to coat the stent morbidity and mortality. or graft according to this invention may be prepared from a In another embodiment, it is envisioned to use bucindolol variety of materials. A primary requirement for the matrix is in combination with other therapeutic modalities. Thus, in that it be sufficiently elastic and flexible to remain unruptured addition to the therapies described above, one may also pro on the exposed surfaces of the stent or synthetic graft. 30 C. Dosages vide to the patient more “standard pharmaceutical cardiac The amount of bucindolol that is administered or pre therapies. Examples of other therapies include, without limi scribed to the patient can be about, at least about, or at most tation, other beta blockers, anti-hypertensives, cardiotonics, about 0.1, 0.5, 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, anti-thrombotics, vasodilators, hormone antagonists, ion 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 35 tropes, diuretics, endothelin antagonists, calcium channel 34,35,36, 37,38, 39, 40, 41,42, 43,44, 45,46, 47, 48,49, 50, blockers, phosphodiesterase inhibitors, ACE inhibitors, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62,63, 64, 65,66, 67, angiotensin type 2 antagonists and cytokine blockers/inhibi 68, 69,70, 71,72, 73,74, 75,76, 77,78, 79,80, 81, 82, 83, 84, tors, and HDAC inhibitors. 85, 86, 87, 88, 89,90,91, 92,93, 94, 95, 96, 97,98, 99, 100, Combinations may beachieved by contacting cardiac cells 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 40 with a single composition or pharmacological formulation 230, 240, 250, 260, 270, 280,290, 300, 310,320, 330, 340, that includes both agents, or by contacting the cell with two 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 441, 450, distinct compositions or formulations, at the same time, 460, 470, 480, 490, 500 mg. or any range derivable therein. wherein one composition includes the expression construct Alternatively, the amount administered or prescribed may be and the other includes the agent. Alternatively, the therapy about, at least about, or at most about 0.001, 0.002, 0.003, 45 using bucindolol may precede or follow administration of the 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, other agent(s) by intervals ranging from minutes to weeks. In 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4,0.5,0.6, embodiments where the other agent and expression construct 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, are applied separately to the cell, one would generally ensure 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, that a significant period of time did not expire between the 3.5, 3.6, 3.7.3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 50 time of each delivery, such that the agent and expression 4.9, 5.0 mg/kg, or any range derivable therein, with respect to the weight of the patient. construct would still be able to exert an advantageously com When provided in a discrete amount, each intake of bucin bined effect on the cell. In such instances, it is contemplated dolol can be considered a "dose.” A medical practitioner may that one would typically contact the cell with both modalities prescribe or administer multiple doses of bucindolol over a 55 within about 12-24 hours of each other and, more preferably, particular time course (treatment regimen) or indefinitely. It is within about 6-12 hours of each other, with a delay time of contemplated that bucindolol only about 12 hours being most preferred. In some situations, Bucindolol may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, it may be desirable to extend the time period for treatment 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or significantly, however, where several days (2, 3, 4, 5, 6 or 7) more times or any range derivable therein. It is further con 60 to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the templated that the drug may be taken for an indefinite period respective administrations. of time or for as long as the patient exhibits symptoms of the It also is conceivable that more than one administration of medical condition for which bucindolol was prescribed or either bucindolol, or the other agent will be desired. In this administered. Also, the drug may be administered every 2, 3, regard, various combinations may be employed. By way of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 65 illustration, where the bucindolol is “A” and the other agent is 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, “B”, the following permutations based on 3 and 4 total admin 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 istrations are exemplary: US 8,080,578 B2 55 56 vi. Miscellaneous Antihyperlipoproteinemics Non-limiting examples of miscellaneous antihyperlipo ABABABBBAAAABBAAABBBBBABBAB AAB.BABABABB, ABBAAB, ABABAABBBBA proteinemics include acifran, azacosterol, benfluorex, B-ben AAABBAAAABAAAABAABBBBABBBBAB Zalbutyramide, carnitine, chondroitin Sulfate, clomestrone, detaXtran, dextran Sulfate Sodium, 5.8, 11, 14, 17-eicosapen Other combinations are likewise contemplated. taenoic acid, eritadenine, furazabol, meglutol, melinamide, 1. Pharmacological Therapeutic Agents mytatrienediol, ornithine, Y-ory Zanol, pantethine, pentaeryth Pharmacological therapeutic agents and methods of ritol tetraacetate, C-phenylbutyramide, pirozadil, probucol (lorelco), B-sitosterol, Sultosilic acid-piperazine salt, tiad administration, dosages, etc., are well known to those of skill 10 in the art (see for example, the “Physicians Desk Reference'. enol, triparanol and Xenbucin. Klaassen’s “The Pharmacological Basis of Therapeutics'. b. Antiarteriosclerotics “Remington’s Pharmaceutical Sciences', and “The Merck Non-limiting examples of an antiarteriosclerotic include Index, Eleventh Edition', incorporated herein by reference in pyridinol carbamate. 15 c. Antithrombotic/Fibrinolytic Agents relevant parts), and may be combined with the invention in In certain embodiments, administration of an agent that light of the disclosures herein. Some variation in dosage will aids in the removal or prevention of blood clots may be necessarily occur depending on the condition of the Subject combined with administration of a modulator, particularly in being treated. The person responsible for administration will, treatment of athersclerosis and vasculature (e.g., arterial) in any event, determine the appropriate dose for the individual blockages. Non-limiting examples of antithrombotic and/or Subject, and Such individual determinations are within the fibrinolytic agents include anticoagulants, anticoagulant skill of those of ordinary skill in the art. antagonists, antiplatelet agents, thrombolytic agents, throm Non-limiting examples of a pharmacological therapeutic bolytic agent antagonists or combinations thereof. agent that may be used in the present invention include an In certain aspects, antithrombotic agents that can be admin antihyperlipoproteinemic agent, an antiarteriosclerotic agent, 25 istered orally, such as, for example, aspirin and wafarin (cou an antithrombotic/fibrinolytic agent, a blood coagulant, an madin), are preferred. antiarrhythmic agent, an antihypertensive agent, a vasopres i. Anticoagulants Sor, a treatmentagent for congestive heart failure, an antiangi A non-limiting example of an anticoagulant include aceno nal agent, an antibacterial agent or a combination thereof. coumarol, ancrod, anisindione, bromindione, clorindione, In addition, it should be noted that any of the following may 30 coumetarol, cyclocumarol, dextran Sulfate sodium, dicuma be used to develop new sets of cardiac therapy target genes as rol, diphenadione, ethyl biscoumacetate, ethylidene dicou B-blockers were used in the present examples (see below). marol, fluindione, heparin, hirudin, lyapolate sodium, While it is expected that many of these genes may overlap, oxazidione, pentosan polysulfate, phenindione, phenprocou new gene targets likely can be developed. mon, phosvitin, picotamide, tioclomarol and warfarin. a. Antihyperlipoproteinemics 35 ii. Antiplatelet Agents In certain embodiments, administration of an agent that Non-limiting examples of antiplatelet agents include aspi lowers the concentration of one of more blood lipids and/or rin, a dextran, dipyridamole (persantin), heparin, Sulfinpyra lipoproteins, known hereinas an “antihyperlipoproteinemic.” none (anturane) and ticlopidine (ticlid). may be combined with a cardiovascular therapy according to iii. Thrombolytic Agents the present invention, particularly in treatment of atherscle 40 Non-limiting examples of thrombolytic agents include tis rosis and thickenings or blockages of vascular tissues. In Sue plaminogen activator (activase), plasmin, pro-urokinase, certain aspects, an antihyperlipoproteinemic agent may com urokinase (abbokinase) streptokinase (streptase), anistre prise an aryloxyalkanoic/fibric acid derivative, a resin/bile plase/APSAC (eminase). acid sequesterant, a HMG CoA reductase inhibitor, a nico d. Blood Coagulants tinic acid derivative, a thyroid hormone or thyroid hormone 45 In certain embodiments wherein a patient is suffering from analog, a miscellaneous agent or a combination thereof a hemmorage or an increased likelyhood of hemmoraging, an i. Aryloxyalkanoic Acid/Fibric Acid Derivatives agent that may enhance blood coagulation may be used. Non Non-limiting examples of aryloxyalkanoic/fibric acid limiting examples of a blood coagulation promoting agent derivatives include beclobrate, enzafibrate, binifibrate, include thrombolytic agent antagonists and anticoagulant ciprofibrate, clinofibrate, clofibrate (atromide-S), clofibric 50 antagonists. acid, etofibrate, fenofibrate, gemfibrozil (lobid), nicofibrate, i. Anticoagulant Antagonists pirifibrate, ronifibrate, simfibrate and theofibrate. Non-limiting examples of anticoagulant antagonists ii. Resins/Bile Acid Sequesterants include protamine and vitamine K1. Non-limiting examples of resins/bile acid sequesterants ii. Thrombolytic Agent Antagonists and Antithrombotics include cholestyramine (cholybar, questran), colestipol 55 Non-limiting examples of thrombolytic agent antagonists (colestid) and polidexide. include amiocaproic acid (amicar) and tranexamic acid (am iii. HMG CoA Reductase Inhibitors stat). Non-limiting examples of antithrombotics include Non-limiting examples of HMG CoA reductase inhibitors anagrelide, argatroban, cilstaZol, daltroban, defibrotide, include lovastatin (mevacor), pravastatin (pravochol) or sim enoxaparin, fraxiparine, indobufen, lamoparan, OZagrel, vastatin (Zocor). 60 picotamide, plafibride, tedelparin, ticlopidine and triflusal. iv. Nicotinic Acid Derivatives e. Antiarrhythmic Agents Non-limiting examples of nicotinic acid derivatives Non-limiting examples of antiarrhythmic agents include include nicotinate, acepimox, niceritrol, nicoclonate, nico Class I antiarrythmic agents (sodium channel blockers), mol and oxiniacic acid. Class II antiarrythmic agents (beta-adrenergic blockers), V. Thyroid Hormones and Analogs 65 Class II antiarrythmic agents (repolarization prolonging Non-limiting examples of thyroid hormones and analogs drugs), Class IV antiarrhythmic agents (calcium channel thereof include etoroXate, thyropropic acid and thyroxine. blockers) and miscellaneous antiarrythmic agents. US 8,080,578 B2 57 58 i. Sodium Channel Blockers ergoloid mesylates, fenspiride, indoramin, labetalol, nicer Non-limiting examples of sodium channel blockers goline, prazosin, teraZosin, tolaZoline, trimaZosin and yohim include Class IA, Class IB and Class IC antiarrhythmic bine. In certain embodiments, an alpha blocker may comprise agents. Non-limiting examples of Class IA antiarrhythmic a quinazoline derivative. Non-limiting examples of quinaZo agents include disppyramide (norpace), procainamide (pron 5 line derivatives include alfuZosin, bunaZosin, doxazosin, pra estyl) and quinidine (quinidex). Non-limiting examples of Zosin, teraZosin and trimaZosin. Class IB antiarrhythmic agents include lidocaine (Xylocalne), ii. Alpha/Beta Blockers tocamide (tonocard) and mexiletine (mexitil). Non-limiting In certain embodiments, an antihypertensive agent is both examples of Class IC antiarrhythmic agents include encam an alpha and beta adrenergic antagonist. Non-limiting ide (enkaid) and flecamide (tambocor). 10 ii. Beta Blockers examples of an alpha/beta blocker comprise labetalol (nor Non-limiting examples of a beta blocker, otherwise known modyne, trandate). as a B-adrenergic blocker, a B-adrenergic antagonistora Class iii. Anti-Angiotension II Agents II antiarrhythmic agent, include acebutolol (sectral), alpre Non-limiting examples of anti-angiotension II agents nolol, amoSulalol, arotinolol, atenolol, befunolol, betaxolol. 15 include include angiotensin converting enzyme inhibitors bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol. and angiotension II receptor antagonists. Non-limiting bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, examples of angiotension converting enzyme inhibitors butofilolol, carazolol, carteolol, carvedilol, celiprolol, ceta (ACE inhibitors) include alacepril, enalapril (vasotec), cap molol, cloranolol, dilevalol, epanolol, esmolol (brevibloc), topril, cilaZapril, delapril, enalaprilat, fosinopril, lisinopril, indenolol, labetalol, levobunolol, mepindolol, metipranolol, moveltopril, perindopril, quinapril and ramipril. Non-limit metoprolol, moprolol, nadolol, nadoxolol, nifenalol, ing examples of an angiotensin II receptor blocker, also nipradillol, Oxprenolol, penbutolol, pindolol, practolol, prone known as an angiotension II receptor antagonist, an ANG thalol, propanolol (inderal), Sotalol (betapace), Sulfinalol, receptor blocker or an ANG-II type-1 receptor blocker talinolol, tertatolol, timolol, toliprolol and xibinolol. In cer (ARBS), include angiocandesartan, eprosartan, irbesartan, tain aspects, the beta blocker comprises an aryloxypropano 25 losartan and Valsartan. lamine derivative. Non-limiting examples of aryloxypro iv. Sympatholytics panolamine derivatives include acebutolol, alprenolol. Non-limiting examples of a sympatholytic include a cen arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopin trally acting sympatholytic or a peripherially acting sym dolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol, patholytic. Non-limiting examples of a centrally acting sym celiprolol, cetamolol, epanolol, indenolol, mepindolol. 30 patholytic, also known as an central nervous system (CNS) metipranolol, metoprolol, moprolol, nadolol, nipradillol, sympatholytic, include clonidine (catapres), guanabenz oXprenolol, penbutolol, pindolol, propanolol, talinolol, terta (wytensin) guanfacine (tenex) and methyldopa (aldomet). tolol, timolol and toliprolol. Non-limiting examples of a peripherally acting sym iii. Repolarization Prolonging Agents patholytic include a ganglion blocking agent, an adrenergic Non-limiting examples of an agent that prolong repolar 35 neuron blocking agent, a B-adrenergic blocking agent or a ization, also known as a Class III antiarrhythmic agent, alpha1-adrenergic blocking agent. Non-limiting examples of include amiodarone (cordarone) and Sotalol (betapace). a ganglion blocking agent include mecamylamine (inversine) iv. Calcium Channel Blockers/Antagonist and trimethaphan (arfonad). Non-limiting of an adrenergic Non-limiting examples of a calcium channel blocker, oth neuron blocking agent include guanethidine (ismelin) and erwise known as a Class IV antiarrythmic agent, include an 40 reserpine (serpasil). Non-limiting examples of a B-adrenergic arylalkylamine (e.g., bepridile, diltiazem, fendiline, gallo blocker include acenitolol (sectral), atenolol (tenormin), pamil, prenylamine, terodiline, Verapamil), a dihydropyri betaxolol (kerlone), carteolol (cartrol), labetalol (normodyne, dine derivative (felodipine, isradipine, nicardipine, nife trandate), metoprolol (lopressor), nadanol (corgard), penb dipine, nimodipine, nisoldipine, nitrendipine) a piperaZinde utolol (levatol), pindolol (visken), propranolol (inderal) and derivative (e.g., cinnarizine, flunarizine, lidoflazine) or a 45 timolol (blocadren). Non-limiting examples of alpha1-adren micellaneous calcium channel blocker Such as bencyclane, ergic blocker include praZosin (minipress), doxazocin (car etafenone, magnesium, mibefradil or perhexyline. In certain dura) and teraZosin (hytrin). embodiments a calcium channel blocker comprises a long V. Vasodilators acting dihydropyridine (nifedipine-type) calcium antagonist. In certain embodiments a cardiovasculator therapeutic V. Miscellaneous Antiarrhythmic Agents 50 agent may comprise a vasodilator (e.g., a cerebral vasodilator, Non-limiting examples of miscellaneous antiarrhymic a coronary vasodilator or a peripheral vasodilator). In certain agents include adenosine (adenocard), digoxin (lanoxin), preferred embodiments, a vasodilator comprises a coronary acecamide, ajmaline, amoproxan, aprindine, bretylium tosy vasodilator. Non-limiting examples of a coronary vasodilator late, bunaftine, butobendine, capobenic acid, cifenline, dis include amotriphene, bendazol, benfurodil hemisuccinate, opyramide, hydroquinidine, indecamide, ipatropium bro 55 benZiodarone, chloracizine, chromonar, clobenfurol, cloni mide, lidocaine, lorajmine, lorcamide, meobentine, trate, dilaZep, dipyridamole, droprenilamine, efloxate, eryth moricizine, pirmenol, prajmaline, propafenone, pyrinoline, rity1 tetranitrane, etafenone, fendiline, floredil, ganglefene, quinidine polygalacturonate, quinidine Sulfate and Viquidil. herestrol bis(B-diethylaminoethyl ether), hexobendine, itra f. Antihypertensive Agents min tosylate, khellin, lidoflanine, mannitol hexanitrane, Non-limiting examples of antihypertensive agents include 60 medibazine, nicorglycerin, pentaerythritol tetranitrate, pen sympatholytic, alpha/beta blockers, alpha blockers, anti-an trinitrol, perhexyline, pimethylline, trapidil, tricromyl, tri giotensin II agents, beta blockers, calcium channel blockers, metazidine, trolnitrate phosphate and Visnadine. vasodilators and miscellaneous antihypertensives. In certain aspects, a vasodilator may comprise a chronic i. Alpha Blockers therapy vasodilator or a hypertensive emergency vasodilator. Non-limiting examples of an alpha blocker, also known as 65 Non-limiting examples of a chronic therapy vasodilator an C.-adrenergic blocker or an O-adrenergic antagonist, include hydralazine (apresoline) and minoxidil (loniten). include amoSulalol, arotinolol, dapiprazole, doxazosin, Non-limiting examples of a hypertensive emergency vasodi US 8,080,578 B2 59 60 lator include nitroprusside (nipride), diaZoxide (hyperstat tensin amide, dimetofrine, dopamine, etifelmin, etillefrin, IV), hydralazine (apresoline), minoxidil (loniten) and Vera gepefrine, metaraminol, midodrine, norepinephrine, pamil. pholedrine and synephrine. vi. Miscellaneous Antihypertensives g. Treatment Agents for Congestive Heart Failure Non-limiting examples of miscellaneous antihyperten Non-limiting examples of agents for the treatment of con sives include ajmaline, y-aminobutyric acid, bufeniode, cicle gestive heart failure include anti-angiotension II agents, after tainine, ciclosidomine, a cryptenamine tannate, fenoldopam, load-preload reduction treatment, diuretics and inotropic floseduinan, ketanserin, mebutamate, mecamylamine, meth agents. yldopa, methyl 4-pyridyl ketone thiosemicarbazone, i. Afterload-Preload Reduction muZolimine, pargyline, pempidine, pinacidil, piperoxan, pri 10 maperone, a protoveratrine, raubasine, rescimetol, ril In certain embodiments, an animal patient that can not menidene, Saralasin, sodium nitrorusside, ticrynafen, tri tolerate an angiotension antagonist may be treated with a methaphan camsylate, tyrosinase and urapidil. combination therapy. Such therapy may combine administra In certain aspects, an antihypertensive may comprise an tion of hydralazine (apresoline) and isosorbide dinitrate (isor arylethanolamine derivative, a benzothiadiazine derivative, a 15 dil, sorbitrate). N-carboxyalkyl (peptide/lactam) derivative, a dihydropyri ii. Diuretics dine derivative, a guanidine derivative, a hydrazines/phthala Non-limiting examples of a diuretic include a thiazide or Zine, an imidazole derivative, a quanternary ammonium com benzothiadiazine derivative (e.g., althiazide, bendroflumet pound, a reserpine derivative or a Suflonamide derivative. hazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, Arylethanolamine Derivatives. Non-limiting examples of chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthi arylethanolamine derivatives include amosulalol, bufuralol, azide, epithiazide, ethiazide, ethiazide, fenguizone, hydro dilevalol, labetalol, pronethalol, Sotalol and sulfinalol. chlorothiazide, hydroflumethiazide, methyclothiazide, meti Benzothiadiazine Derivatives. Non-limiting examples of Crane, metolaZone, paraflutizide, polythizide, benzothiadiazine derivatives include althizide, bendroflume tetrachloromethiazide, trichlormethiazide), an organomercu thiazide, benzthiazide, benzylhydrochlorothiazide, buthiaz 25 rial (e.g., chlonnerodrin, meralluride, mercamphamide, mer ide, chlorothiazide, chlorthalidone, cyclopenthiazide, captomerin Sodium, mercumallylic acid, mercumatilin cyclothiazide, diazoxide, epithiazide, ethiazide, fenduizone, dodium, mercurous chloride, mersalyl), a pteridine (e.g., fur hydrochlorothizide, hydroflumethizide, methyclothiazide, therene, triamterene), purines (e.g., acefylline, 7-morpholi meticrane, metolaZone, paraflutizide, polythizide, tetrachlo nomethyltheophylline, pamobrom, protheobromine, theo rmethiazide and trichlormethiazide. 30 bromine), steroids including aldosterone antagonists (e.g., N-carboxyalkyl (peptide/lactam) Derivatives. Non-limit canrenone, oleandrin, spironolactone), a Sulfonamide deriva ing examples of N-carboxyalkyl (peptideflactam) derivatives tive (e.g., acetazolamide, ambuside, azosemide, bumetanide, include alacepril, captopril, cilaZapril, delapril, enalapril, butazolamide, chloraminophenamide, clofenamide, clopam enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, ide, clorexolone, diphenylmethane-4,4'-disulfonamide, dis quinapril and ramipril. 35 ulfamide, ethoXZolamide, furosemide, indapamide, mefru Dihydropyridine Derivatives. Non-limiting examples of side, methazolamide, piretanide, quinethaZone, torasemide, dihydropyridine derivatives include amlodipine, felodipine, tripamide, Xipamide), a uracil (e.g., aminometradine, ami isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine Sometradine), a potassium sparing antagonist (e.g., and nitrendipine. amiloride, triamterene) or a miscellaneous diuretic Such as Guanidine Derivatives. Non-limiting examples of guani 40 aminozine, arbutin, chlorazanil, ethacrynic acid, etoZolin, dine derivatives include bethanidine, debrisoquin, guana hydracarbazine, isosorbide, mannitol, metochalcone, benz, guanacline, guanadrel, guanaZodine, guanethidine, muZolimine, perhexyline, ticrnafen and urea. guanfacine, guanochlor, guanoXabenz, and guanoxan. iii. Inotropic Agents Hydrazines/Phthalazines. Non-limiting examples of Non-limiting examples of a positive inotropic agent, also hydrazines/phthalazines include budralazine, cadralazine, 45 known as a cardiotonic, include acefylline, an acetyldigi dihydralazine, endralazine, hydracarbazine, hydralazine, toxin, 2-amino-4-picoline, aminone, benfurodil hemisucci pheniprazine, pildralazine and todralazine. nate, bucladesine, cerberosine, camphotamide, convalla Imidazole Derivatives. Non-limiting examples of imida toxin, cymarin, denopamine, deslanoside, digitalin, digitalis, Zole derivatives include clonidine, lofexidine, phentolamine, digitoxin, digoxin, dobutamine, dopamine, dopexamine, tiamenidine and tolonidine. 50 enoXimone, erythrophleine, fenalcomine, gitalin, gitoxin, Quanternary Ammonium Compounds. Non-limiting glycocyamine, heptaminol, hydrastinine, ibopamine, a lana examples of quanternary ammonium compounds include toside, metamivam, milrinone, nerifolin, oleandrin, ouabain, aZamethonium bromide, chlorisondamine chloride, hexam oxyfedrine, prenalterol, proscillaridine, resilbufogenin, Scil ethonium, pentacynium bis(methylsulfate), pentamethonium laren, Scillarenin, strphanthin, Sulmazole, theobromine and bromide, pentolinium tartrate, phenactropinium chloride and 55 Xamoterol. trimethidinium methosulfate. In particular aspects, an intropic agent is a cardiac glyco Reserpine Derivatives. Non-limiting examples of reserpine side, a beta-adrenergic agonist or a phosphodiesterase inhibi derivatives include bietaserpine, deserpidine, rescinnamine, tor. Non-limiting examples of a cardiac glycoside includes reserpine and Syrosingopine. digoxin (lanoxin) and digitoxin (crystodigin). Non-limiting Suflonamide Derivatives. Non-limiting examples of sul 60 examples of a B-adrenergic agonist include albuterol, bam fonamide derivatives include ambuside, clopamide, furo buterol, bitolterol, carbuterol, clenbuterol, clorprenaline, semide, indapamide, quinethaZone, tripamide and Xipamide. denopamine, dioxethedrine, dobutamine (dobutrex), dopam vii. Vasopressors ine (intropin), dopexamine, ephedrine, etafedrine, ethylnore Vasopressors generally are used to increase blood pressure pinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, during shock, which may occur during a Surgical procedure. 65 isoetharine, isoproterenol, mabuterol, metaproterenol, meth Non-limiting examples of a vasopressor, also known as an oxyphenamine, oxyfedrine, pirbuterol, procaterol, protoky antihypotensive, include amezinium methyl sulfate, angio lol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tre US 8,080,578 B2 61 62 toquinol, tulobuterol and Xamoterol. Non-limiting examples sents the primary phenotypes of the two receptors (Mason et of a phosphodiesterase inhibitor include aminone (inocor). al., 1999). Despite the substantially greater degree of norepi iv. Antianginal Agents nephrine-mediated stimulation of the Arg,389 receptor, bucin Antianginal agents may comprise organonitrates, calcium dolol effectively antagonized the response. The difference in channel blockers, beta blockers and combinations thereof. the absolute decrease in cAMP production afforded by bucin Non-limiting examples of organonitrates, also known as dolol was greater for cells expressing B1-Arg389: bucindolol nitrovasodilators, include nitroglycerin (nitro-bid, nitrostat), caused a maximal decrease of 435+80 fimol/ml cAMP in isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (as Arg389 cells compared to 115+23 fmol/ml cAMP in Gly389 pirol, vaporole). cells (P<0.008, N=4). The potency of bucindolol was not 2. Surgical Therapeutic Agents 10 found to be different for the response (ED50–46+4.5 and In certain aspects, the secondary therapeutic agent may 35+11 nM, respectively, P=0.94, N=4). In additional experi comprise a Surgery of Some type, which includes, for ments bucindolol alone at concentrations up to 10 LM caused example, preventative, diagnostic or staging, curative and no stimulation of cAMP in cells expressing either receptor palliative Surgery. Surgery, and in particular a curative Sur variant (data not shown). These results thus indicated that the gery, may be used in conjunction with other therapies, such as 15 Arg389 receptor may provide for a greater clinical response the present invention and one or more other agents. to bucindolol in heart failure treatment. Such Surgical therapeutic agents for vascular and cardio vascular diseases and disorders are well known to those of Example 2 skill in the art, and may comprise, but are not limited to, performing Surgery on an organism, providing a cardiovas Response to B-Blockade in Transgenic Mice cular mechanical prostheses, angioplasty, coronary artery reperfusion, catheter ablation, providing an implantable car Using the C-myosin heavy chain promoter, transgenic dioverter defibrillator to the subject, mechanical circulatory mice with targeted ventricular expression of the human B1AR Support or a combination thereof. Non-limiting examples of a (Arg389 or Gly389 forms) were utilized to ascertain allele mechanical circulatory Support that may be used in the 25 specific responses to chronic administration of the B-blocker present invention comprise an intra-aortic balloon counter propranolol. Expression levels of the two receptors were pulsation, left ventricular assist device or combination equivalent. The generation of these mice and their partial thereof. characterization has recently been reported in detail else where (Perez et al., 2003). For the current studies, 3-month IV. Examples 30 old mice of both genotypes, as well as nontransgenic mice, were treated with propranolol (0.5 mg/ml) in their drinking The following examples are included to demonstrate pre water, or water without propranolol (control) continuously ferred embodiments of the invention. It should be appreciated for 6 months. Hearts were then removed and ventricular pro by those of skill in the art that the techniques disclosed in the tein extracts prepared. These were subjected to Western blot examples which follow represent techniques discovered by 35 ting to ascertain expression of the following proteins using the inventor to function well in the practice of the invention, methods previously described (Perez et al., 2003): GOLs, and thus can be considered to constitute preferred modes for Goi2, G-protein coupled receptor kinase-2 (GRK2), adenylyl its practice. However, those of skill in the art should, in light cyclase type 5 (AC5), total phospholamban (T-PLN), phos of the present disclosure, appreciate that many changes can be phorylated phospholamban (P-PLN) and sarcoplasmic endo made in the specific embodiments which are disclosed and 40 plasmic reticulum calcium ATPase-2A (SERCA). Treatment still obtain a like or similar result without departing from the effect was assessed by comparing expression of the proteins spirit and scope of the invention. of untreated and propranolol treated mice, within genotype, by ANOVA. The study was approved by the University of Example 1 Cincinnati Animal Use and Care Committee. 45 As recently reported (Perez et al., 2003), transgenic mice Transfected Cells with targeted expression of B1-Gly389 or B1-Arg389 to the heart exhibit multiple alterations over time (observed as early A. Methods as 6-months of age), in the expression of certain cardiac Chinese hamster fibroblasts (CHW cells) were stably signaling and Ca++ handling proteins. To assess the potential transfected with the human Arg389 and Gly389 cDNAs as 50 for a genotype-specific response to long-term B-blockade, 3 previously described (Mason et al., 1999). Lines with equiva month-old mice expressing each B1AR genotype were lent levels of expression as determined by radioligand bind treated with placebo or the B-blocker propranolol for six ing were studied to ascertain the antagonist effect of bucin months, the Ventricles removed and protein extracts prepared. dolol on norepinephrine stimulated cAMP accumulation. Western blots were utilized to quantitate expression of the Cells in monolayers were treated with 10 uM norepinephrine 55 indicated proteins, comparing the changes in expression by in the absence and presence of various concentrations of treatment within B1AR genotype groups. As shown in FIG. 4. bucindolol for 20 min at 37° C. and HIcAMP isolated by propranolol treatment had no effect (P=0.67) on expression of column chromatography (Salomon, 1991). the indicated proteins in hearts from Gly389 mice. In con B. Results: Response to Bucindolol in Transfected Cells trast, an overall treatment response (either increases or Expression levels in CHW cells of the receptors for the 60 decreases in expression) was observed with propranolol treat functional antagonism studies were 123-19 and 137+16 ment inhearts from Arg389 mice (P<0.002). The directions of fmol/mg for Arg389 and Gly389 cell lines, respectively. Cells these trends induced by B-blockade, which included were exposed to 10 uM of the agonist norepinephrine, in the increases in Gots, P-PLN and T-PLN, and decreases in Goi absence or presence of varying concentrations ofbucindolol. and GRK2, are all considered restorative biochemical and cAMP levels determined. As shown in FIG. 3, Arg389 65 responses in the context of the hypertrophied/failing heart displayed a greater cAMP stimulation to norepinephrine in (Liggett, 2001). Taken together, then, the protein expression the absence of bucindolol compared to Gly389, which repre profiles associated with chronic B-blockade in this transgenic US 8,080,578 B2 63 64 mouse model Suggest that a more favorable response to the influence f-blocker efficacy. Continuous clinical variables B-blocker bucindolol might be expected in B1-Arg389 heart are reported as meantSD, and comparisons were by t-test or failure patients compared to those with the B1-Gly389 geno Wilcoxon rank-Sum tests. Categorical variables are reported type. as proportions and comparisons were by chi-square or Fish er's exact tests. Cumulative survival curves were constructed Example 3 by Kaplan-Meier methods (Kaplanet al., 1958); and SAS (r) proprietary software, release 6.12. Cary, N.C.: SAS Institute, Bucindolol Vs. Placebo Clinical Study 1996). The Cox proportional-hazards regression model was used to examine the effects of treatment stratified by the A. Materials and Methods 10 indicated genotype. Results were adjusted for age, sex, race, 1. Patient Population and, as indicated, the B1-Gly49 polymorphism. Because of Patients who participated in the Beta Blocker Evaluation of the limited number of comparisons, and an a priori hypothesis Survival Trial (BEST), (Lowes et al., 2002) and who con that was based on cell, transgenic, and other human studies sented for DNA substudies, were genotyped at the coding (Kaplan et al., 1958); and SAS (r) proprietary software, B1AR polymorphic sites. Enrollment in BEST was from May 15 release 6.12. Cary, N. C.: SAS Institute, 1996: Perez et al., 31, 1995 to Dec. 31, 1998; the study design has been 2003); and Wagoner et al., 2002), the P values <0.05 were described in detail elsewhere (BEST Trial Investigators, considered to as significant, without adjustments for multiple 2001; and Lowes et al., 2002). Briefly, the study was a ran comparisons. domized, multicenter, placebo-controlled trial of the 3rd gen B. Results eration, nonselective B-blocker-vasodilator bucindolol (Bris The results from the transfected cells and transgenic mice tow, 2000) in 2708 patients with Class III/IV heart failure prompted genotyping patients from BEST, a trial of the (BEST Trial Investigators, 2001). Those receiving the active B3-blocker bucindolol in the treatment of Class III-IV heart drug were administered 3 mg bucindolol twice daily for the failure which included a placebo arm (BEST Trial Investiga first week, and up-titrated as tolerated on a weekly basis to 50 tors, 2001). Most demographic and baseline clinical charac mg twice daily (or 100 mg twice daily for patients weighing 25 teristics were not statistically different between those who >75 kg). Of the 2708 patients, 1040 consented for the sub participated in the DNA substudy compared to those who did study and had adequate DNA prepared from a blood sample. not. Of particular note, age, sex, NYHA class and heart failure The study was approved by the BEST DNA Oversight Com etiology were not different. Minor and clinically insignificant mittee and the University of Cincinnati Institutional Review differences between DNA substudy participants vs non-par Board. 30 ticipants were noted in baseline heart rates (-1.3 bpm), sys 2. Genotyping tolic blood pressures (+1.7 mmHg), weight (+1.9 kg), LVEF DNA was extracted from whole blood using standard tech (+0.9%) and percentage of non-whites (-5%). The overall niques (Jones, 1963). Genotyping was performed using meth allele frequency of Arg,389 was 67%, which is similar to the ods exactly as previously described in detail (Small et al., reported allele frequency of this polymorphism in the general 2002). For the B1AR, variations at coding nucleotides 145 35 population (Mason et al., 1999) and in heart failure cohorts and 1165 were delineated, which correspond to amino acids (Smal et al., 2002). 49 and 389. These alleles are designated as B1-Ser49, 131- The characteristics of the patients grouped by the primary Gly49 and B1-Arg,389, B1-Gly389. All the DNA samples hypothesis genotypes (B1AR-389) and treatment, are pro were successfully genotyped at the B1AR-389 locus and 1030 vided in Table 2. There were no differences in age, sex, race, were successfully genotyped at B1AR-49. 40 heart failure etiology, NYHA class, or baseline LVEF 3. Statistical Analysis between groups stratified by placebo, bucindolol treatment, The primary endpoints were all-cause mortality, hospital- or genotype. As can be seen, the number of homozygous izations adjudicated by an endpoints committee (BEST Trial Gly389 individuals was relatively small (52 placebo. 42 Investigators, 2001) to be due to heart failure, and the com- bucindolol). Therefore, Arg homozygotes were compared to bined endpoint of death or heart failure hospitalization. The 45 Gly carriers (those having either one or two Gly alleles). The variants at amino acid position 389 of the B1AR (Arg and four cohorts, grouped by treatment and genotype, then, each Gly) were considered the primary genotypes hypothesized to consisted of >200 subjects (Table 2). TABLE 2 Placebo Group Bucindolol Group N = S25 N = S15 Arg Arg/Gly Gly Arg Arg/Gly Gly homozygous heterozygous homozygous Gly carriers homozygous heterozygous homozygous Gly carriers N = 236 N = 237 N = S2 N = 289 N = 257 N = 216 N = 42 N = 258 Age - 60.5 (11.8) 60.3 (12.4) 59.6 (13.2) 60.2 (12.5) 59.8 (11.8) 61.6 (12.0) 56.6 (14.2) 60.8 (12.5) mean (std) Sex-N (%) Male 187 (79%) 183 (77%) 42 (81%) 225 (78%) 206 (80%) 173 (80%) 34 (81%) 207 (80%) Female 49 (21%) 54 (23%) 10 (19%) 64 (22%) 51 (20%) 43 (20%) 8 (19%) 51 (20%) Race-N (%) Non-black 212 (90%) 182 (77%) 33 (63%) 215 (74%) 215 (84%) 165 (76%) 26 (62%) 191 (74%) Black 24 (10%) 55 (23%) 19 (37%) 74 (26%) 42 (16%) 51 (24%) 16 (38%) 67 (26%) US 8,080,578 B2
TABLE 2-continued Placebo Group Bucindolol Group N = S25 N = S15 Arg Arg/Gly Gly Arg Arg/Gly Gly homozygous heterozygous homozygous Gly carriers homozygous heterozygous homozygous Gly carriers N = 236 N = 237 N = S2 N = 289 N = 257 N = 216 N = 42 N = 258 Etiology - N (%) Schemic 145 (61%) 137 (58%) 34 (65%) 171 (59%) 138 (54%) 129 (60%) 23 (55%) 152 (59%) Non- 91 (39%) 100 (42%) 18 (35%) 118 (41%) 119 (46%) 87 (40%) 19 (45%) 106 (41%) ischemic NYHA Functional Class - N (%) II 223 (94%) 217 (92%) 48 (92%) 265 (92%) 242 (94%) 194 (90%) 36 (86%) 230 (89%) V 13 (6%) 20 (8%) 4 (8%) 24 (8%) 15 (6%) 22 (10%) 6 (14%) 28 (11%) LVEF 90- 23.3 (7.0) 24.2 (7.1) 23.6 (6.8) 24.1 (7.0) 23.4 (7.2) 23.7 (7.1) 22.6 (6.9) 23.5 (7.1) mean (std) Baseline Characteristics of patients in BEST stratified by treatment and genotype. Data are meant SD.
Survival of placebo and bucindolol treated patients strati line predictors of mortality, with Ln norepinephrine associ fied by B1AR-389 genotype is shown in FIGS. 5 and 6. ated with 1.8 and 1.6 fold increases in mortality risk by Individual comparisons adjusted for age, sex, and race 25 univariate and multivariate analyses, respectively. Surpris revealed that homozygous Arg389 bucindolol-treated ingly, as shown below, the change in norepinephrine at 3 patients had increased survival compared to Arg389 placebo months had a complex relationship to mortality that was treated patients (hazard ratio=0.62, 95% CI=0.40 to 0.96, P=0.03). Thus, the improvement in survival due to bucindolol dependent on the treatment group. In the bucindolol-but not in the Arg389 patients amounted to 38% over placebo. This 30 in the placebo-treated group a Substantial number of patients same comparison in Gly carriers revealed no difference in (18% of the subject population) exhibited decreased norepi survival curves (hazard ratio=0.90, 95% CI=0.62 to 1.30, nephrine levels that were associated with a 1.7 fold higher risk P=0.57), indicative of no treatment response to bucindolol. of Subsequent mortality. There was also an apparent influence of B1AR genotype on Most, but not all, studies indicate that adrenergic activity is the heart failure hospitalization response to bucindolol (FIG. 35 a major determinant of outcome in chronic heart failure 5). A decrease in hospitalizations with bucindolol treatment (CHF) (Cohn et al., 1984; Kaye et al., 1995; Isnard et al., in homozygous Arg389 patients was observed compared to 2000; Rockman et al., 1989). In addition, cardiac adrenergic placebo patients with the same genotype (hazard ratio-0.64, activity is the first neurohormonal marker that becomes 95% CI=0.46-0.88, P=0.006). Gly389 carriers showed no elevated in subjects with left ventricular dysfunction (Run benefit of the drug compared to placebo in terms of hospital 40 quist et al., 1997). These observations form the cornerstone of izations (hazard ratio=0.86, 95% CI=0.64 to 1.15, P=0.298). the rationale for B-blocker therapy of heart failure (Bristow, For the combined outcome of heart failure hospitalizations or death, a bucindolol-associated favorable treatment effect 2000). (FIGS. 5 and 7) was evident for Arg389 patients compared to On the other hand, adrenergic Support is an important com placebo (hazard ratio=0.66, 95% CI=0.50 to 0.88, P=0.004), pensatory mechanism in the failing heart, serving to maintain but was not apparent in bucindolol-treated Gly389 carriers vs 45 resting myocardial performance in a relatively normal range placebo (hazard ratio=0.87, 95% CI=0.67 to 1.11, P=0.250). (Port et al., 2001). When adrenergic drive is rapidly reduced Adjustments for the position 49 variants (3-Ser49, in subjects with chronic heart failure myocardial function B1-Gly49) had no significant effect on any of the above may worsen (Gaffney et al., 1963), and treatments which results. With Such adjustment (including age, sex and race) Substantially lower adrenergic drive may increase serious the hazard ratio for survival for B1-Arg389 homozygotes was 50 adverse events including mortality (Cohn et al., 2003; Swed 0.62, 95% CI=0.40 to 0.97, P=0.035; for B1-Gly389 carriers berg et al., 2002). Based on these observations it appears that there remained no apparent treatment effect (hazard “sympatholytic' pharmacological lowering of adrenergic ratio=0.89, 95% CI=0.61 to 1.30, P=0.56). Similarly, adjust activity may affect heart failure natural history quite differ ment for position 49 had no appreciable effect on the hazard ently from B-blockade. ratios for hospitalizations: for Arg389 homozygotes the haz 55 Although baseline adrenergic activity has been examined ard ratio=0.63, 95% CI=0.45 to 0.86, P=0.007; for Gly389 in numerous CHF outcome studies as well as in clinical trials carriers the hazard ratio=0.85, 95% CI=0.63 to 1.14, P=0.54. (Benedict et al., 1996; Swedberg et al., 1996; Francis et al., The results for the combined outcome of death and hospital 1993; Anand et al., 2003), until recently only relatively small izations stratified by B1-389 genotype were also not modified numbers (typically hundreds) of subjects have been investi by the 1-49 genotypes. 60 gated in these studies (Anand et al., 2003). In addition, the Example 4 relationship of temporal behavior of norepinephrine as a potential determinant of natural history has been examined in Mortality Risks Associated with Sympatholysis in only two other trials (Swedberg et al., 1990; Anand et al., Some Best Patients 2003) and never in a large CHF cohort, placebo-controlled 65 study employing a powerful anti-adrenergic agent. Thus, the Systemic venous norepinephrine measurements as part of effects of baseline levels and changes in adrenergic activity the BEST Trial core protocol were among the strongest base on clinical outcomes in the BEST were investigated, as well US 8,080,578 B2 67 68 as and the interaction of bucindolol, a B-blocker with sym (p<0.0001) and 12 (p<0.0001) months. Relative to changes in patholytic properties on clinical outcomes. the placebo group, the decrease in norepinephrine in the A. Methods bucindolol group was by 19% and 13% at 3 and 12 months, 1. Clinical Protocol respectively. The BEST protocol and the main outcomes have been 3. Baseline Norepinephrine as a Predictor of Mortality or previously described (Mason et al., 1999; Small et al., 2002). the Combined Endpoint of Mortality+CHF Hospitalization Because of an initial delay in setting up the procedures, col FIG. 9 plots the hazard ratios for total mortality risk for lection of blood samples for norepinephrine in all randomized baseline norepinephrine values, by quartiles relative to the patients began 6 months after trial initiation. As a result, 2126 first quartile assigned a hazard ratio (HR) of 1.0. For the entire of the 2708 randomized subjects in BEST had at least a 10 cohort and for each treatment group there is a progressive baseline norepinephrine sample collected and measured. increase in mortality risk with increasing quartile. Similar 2. Norepinephrine Sample Collection and Measurements results were obtained for the combined endpoint of mortal Peripheral venous NE samples were drawn at baseline, 3 ity+CHF hospitalization (FIG. 10). and 12 months by inserting a 21 gauge butterfly needle into an Table 3 gives the univariate and multivariate analyses of arm vein and placing the Subject in a quiet room in a Supine 15 baseline norepinephrine and other protocol prespecified position for 30 minutes. The initial 3 ml of blood was dis potential modifiers of mortality. Ln norepinephrine yielded a carded, and then 5 ml of blood was withdrawn and immedi univariate HR (95% confidence limits) of 1.82 (1.58-2.09), ately transferred to pre-chilled 5 ml tubes containing EDTA. p-0.001. On multivariate analysis Ln norepinephrine was Within 30 min plasma was separated and frozen at -70° C. among the most powerful predictors of mortality. Sites shipped samples on dry ice to a central laboratory (Lab 4. Change in Norepinephrine as a Predictor of Mortality or Corp, Raritan, N.J.) every 3 mo, where the samples were the Combined Endpoint of Mortality+CHF Hospitalization stored at -85°C. and assayed within 3 weeks. NE was mea The relationships of quartile changes in norepinephrine at sured by HPLC-electrochemical detection using the Bio-Rad 3 months to subsequent mortality or mortality+CHF hospi HPLC method (Bio-Rad Laboratories Hercules, Calif.). talization are shown in Table 4, where HRs are calculated Quality control included re-measuring all samples with initial 25 relative to the 1st quartile of change. The quartile analysis was values of <200 pg/ml or >2000 pg/ml, from the 2" stored performed in order to keep the norepinephrine change/duar tube; and routinely (every 20 samples) measuring known tile the same in the placebo and bucindolol groups, with the amountS. cut points derived from the entire cohort. This created 2 3. Statistical Methods quartiles of norepinephrine reduction (1' and 2"), and 2 of Means and standard deviations (SD) for continuous data, 30 norepinephrine increase (3" and 4"). Both absolute norepi and proportions or percentages for categorical data are pre nephrine change in pg/ml and % change from baseline value sented. T-tests or Wilcoxon rank sum tests were used for are given in Table 4. Because of the sympatholytic effect of continuous data, and chi-square or Fisher's exact test for bucindolol there were more bucindolol patients in the 1 categorical data. An alpha level of 0.05 (2 tailed, unadjusted) quartile and more placebo patients in the 4" quartile. was used to indicate statistical significance. 35 As can be observed in Table 4, for absolute norepinephrine Norepinephrine levels at baseline, or the change at 3 change vs. mortality the placebo group exhibited a trend for months were used to predict survival and the combined end an increased risk in the 4th/1st quartile, with an HR of 1.38, point of mortality+CHF hospitalization. Absolute and log p=0.099), and no trends for differences in mortality in the 2nd transformed data were initially analyzed. Because of skew or 3rd quartiles relative to the 1st. For mortality+CHF hospi ness in norepinephrine levels natural log(Ln) transformed 40 talization, in the placebo group the 4" quartile:1st had a data were used in multivariate Cox proportional hazards significant HR of 1.46 (p=0.011). In contrast, the bucindolol regression models. group exhibited no trends for an increased risk in the 4":1 A Maximum Likelihood based method (Kalbfleisch et al., quartiles for either clinical outcome, but a decreased risk in 1980) was used to categorize changes in norepinephrine into mortality in the 3rd quartile relative to the 1' (HR 0.66, 3 groups for prediction of mortality or mortality+CHF hos 45 p=0.046) and a trend (p=0.22) for a decreased risk in the pitalization. This partitioning method finds the optimal split 3":1 quartile for mortality+CHF hospitalization. of norepinephrine values that maximize the likelihood of the For norepinephrine '% change there were increases or resulting Cox proportional hazards model. In addition, a flex trends for increases in risk in the placebo 3":1 and 4":1 ible cubic spline analysis (Green et al., 1994) was used to quartiles, for both mortality and mortality+CHF hospitaliza determine the shape and significance level of the relationship 50 tion. In contrast, in the bucindolol group there were no such of norepinephrine changes at 3 months to Survival. trends for an increased HR in the 3" or 4" quartile relative to B. Results the 1 for either clinical endpoint, and similar to the absolute 1. Study Population norepinephrine change there was a trend for a decreased HR The baseline demographic and population descriptor data (0.77) in the 3'1' quartile (p=0.21). in Subjects in whom at least a baseline norepinephrine was 55 Table 4 also gives HRS by treatment group expressed as drawn were not different from the entire study population bucindolol:placebo, for each norepinephrine quartile by (BEST Trial Investigators, 2001). absolute or % change. For mortality, the bucindolol: placebo 2. Norepinephrine Data HR was significantly
Effect of change in norepinephrine (NE) at 3 months on Subsequent mortality (M) or mortality + CHF hospitalization (M+H) in the placebo and bucindolol groups, and treatment effects ofbucindolol compared to placebo by norepinephrine change quartile, hazard ratio and (95% confidence intervals)
Mortality hazard ratios by Crude mortality (%) and NE Change NE change quartile relative bucindolol placebo hazard ratios by Absolute, to 1 quartile NE change quartile
pg/ml 2nd 1st 3rd 1st 4th, 1st 1st 2nd 3rd 4th
Placebo O.98 1.01 1.38 24.5 27.2 27.5 37.5 (P): M (0.65-1.48) (0.68-1.51) (0.94-2.03) M-H 1.21 1.11 1.46 (0.89-1.64) (0.82-1.50) (1.09-196) Bucindolol O.99 O.66 1.15 26.1 25.7 17.7% 28.6 (B): M (0.70-1.41) (0.43-0.99) (0.80-1.65) M-H 1.OO O.83 1.10 (0.75-1.32 (0.61-1.12) (0.82-147) BP: O.96 O.98 O.63 O.8O M (0.65-143) (0.68-143) (0.41-0.96) (0.57-1.14) M-H 1.19 1.30 O.S8 O.74 (0.88-1.61) (0.98-1.72) (0.43-0.78) (0.56-0.98) % Change
Placebo 1.21 1.42 1.37 23.1 27.3 32.1 29.2 (P): M (0.80-1.84) (0.96-2.10) (0.92-2.04) M-H 1.27 1.33 1.35 (0.93-1.72) (0.99-1.78) (1.00- 1.81) Bucindolol 1.14 O.77 1.19 25.2 26.0 18.8 29.1 (B): M (0.80-1.62) (0.51-1.16) (0.83-1.71) M-H 1.18 O.91 1.18 (0.89-1.56) (0.67-1.24) (0.88-1.58) BP:M 1.03 O.98 O.S6 O.90 (0.69-1.54) (0.68-142) (0.38-0.84) 0.63-1.29) M-H 1.14 1.39 O.65 O.66 (0.84-1.54) (1.05-1.85) (0.48-0.88) (0.50-0.88)
Quartiles are: absolute NE change in pg/ml, 1st<-144 (placebo n = 155, bucindolol n = 268); 2nd -144 to <-9 (placebo n = 206, bucindolol n = 214), 3rd-9 to 111 (placebo n = 236, bucindolol n = 186), 4th >111 (placebo in 248, bucindololn = 173); %NEchange, 1st<-30.2(placebo n = 160, bucindolol n = 262), 2nd-30.2 to <-2.5 (placebo n = 198, bucindololn = 223), 3rd-2.5 to 31.1 (placebo n = 240, bucindolol n = 181), 4th >31.1 (placebo n = 247, bucindolol n = 175), p < .05 vs 1st quartile, Fisher's Exact Test US 8,080,578 B2 73 74 TABLE 5 Demographic characteristics of likelihood-determined subgroups with increased risk associated with changes (A) in norepinephrine (NE, in pg/ml) by reductions (Redxn) or increases (Incr) vs. the Intermediate (Inter) NE change Subgroups. NE is in pg/ml. data in meant SD: *, ps.OS vs. Inter. H. p s.10 VS. Inter Placebo Bucindolol Redxn Inter Incr Redxn Inter Incr Parameter (A NE is -783) (A NE > -783, a 362) (A NE > 362) (A NE s -244.5) (A NE-244.5, a +145) (A NE > 145) Number of n = 11 n = 762 n = 72 n = 153 n = 551 n = 137 Subjects Baseline NE, 1500*. 405 464 - 245 514328 932* 544 422, 189 409 - 199 pg/m NE change (c) -1024* 220 -16 188 642* 335 -529* 458 -44 + 103 326 244 3 mos, pg/m NE change (c) -667* S28 45 - 268 297* - 560 -349* 347 17.5 - 216 161* 246 12 mos, pg/m Number of 6 (55%)* 200 (26%) 34 (47%)* 52 (34%)* 114 (21%) 43 (31%)* deaths (%) Age (years) 63.69.8 603 - 11.9 64.7* 10.8 60.1 - 12.2 60.7 - 12.1 62.O. 12.7 Gender (% M/F) 6436 80.2O 82.18 79.21 81,19 82.18 Race (% Non- 73/27 80.2O 82.18 74/26# 80.2O 77/23 Black/Black) NYHA Class 82.18 92.8 83/17% 86/143 93.7 93.7 (% III/IV) Duration of 73. Of 39.0 36.0 36.0 36.0 31.0 CHF, mos, median Etiology (% 45/55 42.58 29.71% 4654 42.58 31,69* Non-ischemic Schemic Baseline Heart 79.2 12.4 816 - 12.8 78.3* 12.3 8S.S* 13.8 81.0 - 13.0 80.6 - 13.7 Rate (HR, BPM) HR change (a) -55 - 17.0 -2.3 - 12.5 1.O* 13.3 -13.6* 14.8 -9.7 12.4 -7.1* 12.8 3 mos HR change (c) -10.7"+ 13.2 -2.6 13.5 -1.8 13.5 -12.6* 14.4 -7.9 - 13.5 -8.1 + 14.6 2 mos Systolic BP 111 20 118 - 18 11619 11619 118 - 18 120 18.2 (SBP, mm Hg) Change in SBP 4.7 13.8 O.O -0.1 - 18.1 -0.7 - 18.2 -0.5 - 15.7 -4.7* 16.4 (c) 3 mos 15.4 Change in SBP 5.6 19.5 O.7 18.1 1921.0 2.7 20.1 O.7 18O -O.9 16.8 (a) 12 mos LVEF, EF 22.8 6.6 23.17.2 23.37.6 20.1* 8.0 24.1 - 7.0 233 6.7 units (EFU) as %) Change in O.2 6.4 2.3 6.6 O.6* 7.2 7.0"E 8.4 S.77.9 42* 7.0 LVEF (a) 3 mos, EFU Change in 5.7; 11.9 3.38.7 1.4 - 7.6 8.89.2 7.3 10.4 7.18.8 LVEF (a) 12 mos, EFU
Example 5 ODel322-325) in BEST, comparison to that originally reported by Liggett’s group (Small et al., 2002). Samples Prevalence of A2C-Adrenergic Receptor Genetic 50 from BEST were evaluated by using primers to amplify by Variants in Best PCR a region of the CAR sequence that covers the deletion The table below gives the prevalence (%) of C-adrenergic and then running the amplification reaction overagel that was receptor genetic variants (WT/WT-homozygous wild type, capable of resolving a 12-base pair difference in length WT/DEL–heterozygotes, DEL/DEL-homozygous between products with or without the deletion. TABLE 6
Non-Black Black Entire Cohort
Study WTWT WTDEL DELDEL WTWT WTDEL DELDEL WTWT WTDEL DELDEL
BEST 91.6 8.2 O.2 33.8 47.8 18.4 80.0 16.1 3.9 Small, et 86.4 6.2 7.4 29.5 S2.6 58.5 11.9 29.6 a CHF Small, 94.3 3.8 1.9 34.5 48.8 16.6 67.7 23.8 8.5 controls US 8,080,578 B2 75 76 As can be seen in the above table, the frequency of the stimulation by a 5-ms pulse at 10% above threshold was then C.2cDel322-325 allele is much greater in Black populations applied, and after equilibration full dose-response curves to vs. non-Black, in BEST 0.423 vs. 0.043 (p<0.0001). Sec isoproterenol, bucindolol or Xamoterol were performed using ondly, in Blacks in BEST the C2cDel322-325 allele fre the indicated concentrations and application of increasing quency is not as high as in Small et al’s Black CHF patients doses every 5 minutes. In experiments in which forskolin was (0.615, p<0.0001), but is similar to that in Smalletal's Black used to enhance signal transduction {e,f, 10 M of this controls (0.411, p=0.85). These differences probably reflect adenylyl cyclase activator was applied to the tissue baths the relatively small sample sizes employed in Small et al.'s 15-20 minutes prior to the performance of dose-response (n=78 Black CHF patient, 84 Black controls) and the BEST curves, and allowed to achieve stability of tension response. trial (n=207 Blacks in the DNA substudy). 10 Systolic tension at each dose was calculated as the stimulated In humans increased adrenergic drive associated with the tension in mN/mm minus baseline tension. The maximum ODel322-325 polymorphism has only been assumed, and tension, concentration of isoproterenol that produced 50% of has not been directly investigated. the maximum developed tension (ECs) and curve slope were One possible reason for the small difference in baseline computed by nonlinear repeated measures analysis of cova norepinephrine between C.2cDel322-325 homozygotes and 15 riance. A statistically significant negative or positive curve C.2c wild type controls that is directly addressed in this pro slope on grouped data was used to identify negative or posi posal, is that systemic venous norepinephrine is not a good tive inotropic effects, respectively, and curve slope differ Surrogate indicator of cardiacadrenergic drive, and in chronic ences between genotypic groups were detected by a test for heart failure changes in cardiac adrenergic drive can occur in interaction. Statistical methods were employed as described the absence of changes insystemic norepinephrine. in Examples 3 and 4. The results of baseline and 3-month change in norepineph 2. Transfected Cells, radioligand binding, cAMP assays rine by C.2c receptor type is shown in Table 7: Chinese hamster fibroblasts were stably transfected using TABLE 7 Norepinephrine (NE), meant SD, and (n) from the BEST Trial, by C.2c Receptor Type: 'ps OS vs. placebo change Ca2c Receptor Baseline NE, Change in NE (pg/ml) at 3 mos Type pg/ml Placebo Bucindolol All C.2c wild type 479 + 264 (710) 12 + 274 (305) -50* + 227 (304) -19 + 254 (609) homozyg. or heterozyg. Ca2cDel322-325 521 + 350 (161) 51 + 323 (60) -153* + 468 (67) –57 + 417 (127) homozygotes
As seen in Table 7, the baseline levels or change in systemic constructs previously described so as to separately express venous NE in BEST strongly support the above-stated the human B-Arg389 or f3-Gly389 receptors. BAR expres hypotheses regarding effects of the C.2cDel322-325 receptor sion, and affinity for bucindolol, were determined by radioli variant on baseline adrenergic drive; there is only a nonsig 40 gand binding studies with 'I-cyanopindolol (I-CYP), nificant trend in favor of the C.2cDel322-325 homozygotes for using 1 uM propranolol to define nonspecific binding as a higher baseline norepinephrine at 3 months. On the other described. Whole cell cAMP accumulation studies were car hand, it can be readily appreciated in Table 7 that the decrease ried out by the H-adenine method using two lines with in norepinephrine with bucindolol is much larger in 45 equivalent expression levels of the two receptors as indicated. C.2cDel322-325 homozygotes than in C.2c wildtype homozy Attached cells were exposed to vehicle (basal), 10 uM nore gotes or heterozygotes. pinephrine or 10 uM norepinephrine with the indicated con centrations of bucindolol for 15 min at 37° C. Example 6 B. Results 50 1. Human Ventricular ExVivo Contractile Responses Cor Expansion of Examples 3 and 4 relate with BAR Genotype In these studies isolated right ventricular trabeculae from A. Materials and Methods human hearts were utilized to ascertain the effects of geno 1. Ex Vivo Human Ventricular Studies type on contraction using the relevant tissue, under endog Nonfailing hearts were obtained from local potential organ 55 enous expression, in the absence and presence of Ventricular donors whose hearts were not transplanted because of physi failure. The pre-explant LVEF in the nonfailing group was cal or ABO blood type incompatibility. Failing hearts were 0.61+0.13 for Arg and 0.53+0.15 for Gly, and in the failing from patients with end-stage heart failure due to ischemic or group=0.21+0.11 for Arg and 0.17+0.07 for Gly. Five of 11 non-ischemic dilated cardiomyopathies who underwent car failing Arg and six of 11 failing Gly patients had ischemic diac transplantation. The demographic characteristics of the 60 dilated cardiomyopathies, with all the other failing hearts hearts are provided in Results. The contractile response of being nonischemic dilated cardiomyopathies. The ages of the isolated, field stimulated, human trabeculae was assessed as nonfailing hearts were: Arg 39-16 years, Gly 43+20 years previously described 1755: a-c). Trabeculae of uniform size (p=0.64). For failing hearts the ages were: Arg 48+15 years, (1 to 2x6 to 8 mm) were mounted in 80 ml muscle bath Gly 54+8 years, p=0.26). The gender distribution in nonfail chambers in Tyrode's solution at pH 7.45 bubbled with 95% 65 ing hearts was 3 males and 8 females in Arg, and 5 males and O-5% CO, at 36°. After equilibration, a tension of 75% of 6 females in Gly. In Failing hearts the gender distribution was Limax was applied to each individual trabeculum. Field 8 males and 3 females in Arg, and 2 males and 9 females in US 8,080,578 B2 77 78 Gly. Shown in FIG. 12 are systolic tension responses to iso dolol was greater for cells expressing fB-Arg389: bucindolol proterenol in right ventricular trabeculae removed from non caused a maximal decrease of 435+80 fimol/ml cAMP in failing and failing human hearts, stratified by the BAR-389 Arg389 cells compared to 115+23 fmol/ml cAMP in Gly389 genotype. In nonfailing hearts, the responses differed cells (P=0.008, n=4). The potency of bucindolol was not between genotypes, with maximal tensions being higher for found to be different for the response (ECso 46+4.5 and B-Arg,389 homozygotes (13+2.5 vs. 5.2t1.4 mN/mm for 35+11 nM, respectively, P=0.94, n=4). In addition, in ''I- f3-Gly389 carriers, P=0.01). Importantly, this same pheno CYP competition binding studies the affinity for bucindolol type, with an even greater allele-specific relative difference, was not different between B-Arg389 (pK-9.6+0.04) and was observed in trabeculae from failing hearts: maximal iso f3-Gly389 receptors (pK-9.6+0.11, n=3). These findings proterenol-stimulated tensions were 9.4:1.9 mN/mm for 10 indicate that bucindolol is capable of antagonizing the |-Arg,389 and 2.4+0.60 mN/mm for B-Gly389 (P=0.008). enhanced response off-Arg389. A second group of 23 failing hearts was used to assess the 3. Mechanism of the Therapeutic Advantage to the Arg389 inotropic effects of bucindolol, the B-AR selective partial Genotype agonist Xamoterol {d}, and isoproterenol in isolated right Increased adrenergic activity, typically identified by ventricular trabeculae. This group's LVEF averaged 15 elevated systemic venous norepinephrine levels, Supports 0.18+0.09, with 10 nonischemic and 13 ischemic dilated car compromised myocardial function in heart failure patients diomyopathies. The average age was 52t11, and there were but contributes to the progression of heart failure {h}. This 20 males and 3 females. Thirteen of the 23 hearts were complex relationship between adrenergic activity and out homozygous for Arg389, while 10 were Gly carriers (all comes was observed in BEST, where an increase in baseline heterozygotes). There were no differences between Arg norepinephrine was independently associated with adverse homozygotes and Gly carriers with respect to LVEF, age, outcomes, but marked withdrawal of adrenergic activation etiology of cardiomyopathy, and gender. In 8 of the 23 hearts was associated with increased mortality {i}. Unlike other bucindolol and Xamoterol experiments were performed; the B-blockers that have been used to treat heart failure, bucin other 15 hearts had either Xamoterol or bucindolol dose dolol has potent sympatholytic properties, and in BEST 18% response curves performed without the other agent. All 23 25 of patients treated with bucindolol exhibited exaggerated hearts had full isoproterenol dose-response curves per norepinephrine lowering at three months associated with a formed. 1.7 fold increased risk of subsequent mortality i, reminis The results of the isoproterenol dose-response between the cent of increased mortality of patients in the MOXCONTrial two genotypic groups was quite similar to results shown in treated with the pure sympatholytic agent moxonidine {j}. FIG. 12. In data not shown, there was a marked difference in 30 Although the increased mortality risk of exaggerated dose-response in favor of the Arg/Arggenotype, with highly symaptholysis is not completely understood, it probably significant (p<0.001) differences in curve slope by test for involves loss of adrenergically-mediated contractility support interaction and a difference in maxima (Arg, XXXX: Gly, yyyy, to the failing heart. Patients who are Arg homozygous could p-0.05). As can be seen in FIG.3, bucindolol alone produced therefore potentially tolerate the loss of norepinephrine sig a negative inotropic effect in both genotypic groups (negative 35 naling better than Gly carriers, because as shown in FIGS. 12 slopes in both, both p values <0.01, nonsignificant test for and 13, even low levels of catecholamine agonist produce an interaction between curve slopes). In the presence of forsko increase in force in Arg homozygotes. The other mechanism lin pretreatment the Arghearts retained a negative curve slope by which Arg homozygotes could gain a therapeutic advan (P<0.05), but the slope of the Gly dose responsecurve was not tage to treatment by bucindolol would be antagonism of a different from 0. (p=0.25). In the absence of forskolin (FIG. 40 greater degree of adverse B-AR signaling, as implied in FIG. 13C), Xamoterol produced a positive inotropic effect in the 3. In order to determine which of these mechanisms may have Arghearts, but a negative inotropic effect in Gly trabeculae accounted for the more favorable therapeutic effect ofbucin (both slope p values <0.05) Xamoterol when applied with dolol in Arg homozygotes vs. Gly carriers, the inventors forskolin pretreatment produced a positive inotropic effect in compared mortality effects by baseline norepinephrine and both genotypic groups (positive curve slopes in both geno 45 by norepinephrine change at 3 months (Table 8). As can be types, p=<0.05), with the Arg/Arg hearts having a greater seen in Table 8, the hazard ratio of Arg homozygotes to Gly inotropic effect that reached significance compared to base carriers for mortality decreases with increasing baseline nore line at Xamoterol doses of 3x10 M and 107 M. pinephrine, Suggesting a progressive advantage to bucin 2. Functional Antagonism of NE Stimulated cAMP in dolol-treated Arg homozygotes with increasing adrenergic Transfected Cells 50 drive. For the change in norepinephrine analysis, the inven For these studies cells expressing equivalent levels (fmol/ tors compared mortality in Arg homozygotes to Gly carriers mg, n=4) of the Arg,389 (123-19) and Gly389 (137+16) in groups previously identified as being at an increased risk human fBARs were utilized. Basal levels of cAMP were for mortality related to a marked reduction (by >244 pg/ml at 72-8.5 and 5.9-9.1 fmolfwell. Initial cAMP accumulation three months of therapy) in norepinephrine, a reference group experiments in the presence of bucindolol up to 10 LM 55 with little or no change in norepinephrine (-244 to 145 pg/ml) showed no evidence for intrinsic sympathomimetic activity not at risk for increased mortality, and a group at increased (USA) at either receptor. To examine functional antagonism, risk for mortality because of an increase in norepinephrine cells were exposed to 10 uM of the agonist norepinephrine, in (by >145 pg/ml). As can be seen in Table 8, there is no the absence or presence of varying concentrations ofbucin advantage to Arg homozygotes in the Subgroup at increased dolol, and cAMP levels determined. As shown in FIG. 13, 60 risk for mortality from exaggerated sympatholysis (Group 1); Arg389 displayed a greater cAMP stimulation to agonist in the hazard ratio of 1.07 indicates a negligible advantage of the absence of bucindolol compared to Gly389, which repre Gly carriers in this group. On the other hand, as for baseline sents the primary phenotypes of the two receptors as noted norepinephrine, there is a decreasing hazard ratio with earlier {998. Despite the substantially greater degree of NE increasing norepinephrine rise at 3 months, to the point that in mediated stimulation of the Arg389 receptor, bucindolol 65 the increasing norepinephrine group the advantage to Arg effectively antagonized the response. The difference in the homozygotes is by a relatively better mortality reduction of absolute decrease in cAMP production afforded by bucin 64% (p=0.08). These norepinephrine change and baseline US 8,080,578 B2 79 80 data indicate that the bucindolol therapeutic advantage for the from this adverse effect: 1) they have the wild type version of Arg homozygous state of the faR is directly related to the the C receptor, which is one of the determinates of norepi degree of adrenergic activity, and not to protection against nephrine release (Bristow 2000); 2) they have the high func symaptholysis. tioning variant of the B receptor, which presumably allows TABLE 8 Bucindolol-treated patients in BEST DNA substudy with norepinephrine (NE) measurements (pg/ml, n = 439 Mortality hazard ratio, Arg, Arg: Gly NE Group carrier 95% C.I. Cox p value # Events BSL NE (n) 64-356 (146) O.90 0.37, 2.22 O.82 19 358-545 (144) O.74 0.37, 1.51 O41 31 546-2571 (149) O.68 0.32, 1.47 O.33 29 *Change in NE (a) 3 mos (n) Group 1, <-244 (70) 1.07 0.41, 2.78 O.89 17 Group 2, -244 to 145 (248) O.82 0.43, 1.59 O.S6 36 Group 3, >145 (54) O.36 0.11, 1.15 O.08 14
BSL = baseline. NE change in the bucindolol group at 3 months (mos) was related to subsequent survival outcome; the cutpoints are from the previously-published likelihood analysis from the entire cohort. In that analysis compared to Group 2, Group had a 1.69 fold (p<05) increase in mortality, and Group 3 a 1.65 fold (p<05) increase in mortality, 25 Example 7 them to withstand loss of norepinephrine signaling. Thus, prescreening for the presence of either the wild-type CAR Additional Analysis from Best Trial or BAR-389Arg/Argidentifies patient populations who have 30 a reduction in mortality if respectively 29% (p=0.031) or 38% In chronic heart failure the activation of adrenergic nervous (p=0.030). system has dual, seemingly antithetical consequences (FIG. In the Black subgroup, it appears to be a specific pharma 14). On the one hand, ongoing adrenergic activation provides cogenomic profile present in Substantial numbers of patients important Support to the failing heart, and pharmacologic that led to inefficacy and a trend for adverse outcomes with abolishment of this Support by sympatholytic agents 35 bucindolol treatment. Blacks have a much higher (approxi increases mortality (Bristow et al., 2004; Cohn et al., 2003). mately 10 fold) allele frequency for a loss of function variant On the other hand, chronic B-adrenergic stimulation is cardi of the C. receptor, involving deletion of amino acids 322-325 omyopathic, and B-adrenergic receptor blockade can improve in the 3rd intracytoplasmic loop of the receptor protein (Small the dilated cardiomyopathyphenotype and clinical outcomes. et al., 2000). Such a loss of function in the CAR would be The challenge of anti-adrenergic therapy is to not Substan 40 expected to increase adrenergic drive, as the normal prejunc tially interfere with adrenergic support, while inhibiting the tional adrenergic inhibitory action of C. receptors is compro adverse effects. The strategy of starting with low doses of mised (Brum et al. 2002). Subjects in BEST and in particular reversible, mass action/competitive B-blocking agents has Blacks who were heterozygous or homozygous (CAR been largely Successful in dealing with this delicate balance, Del322-325 carriers) for this gene variant had a trend for an particularly in less advanced heart failure patients. 45 increased systemic norepinephrine at baseline, and had a It is clear that in BEST bucindolol encountered difficulty much greater sympatholytic response to bucindolol (FIG. with balancing the effects of anti-adrenergic therapy, by with 17). Indeed, approximately 61% of Black subjects in BEST drawing Support excessively in Class IV patients (Anderson were CAR-Del322-325 carriers, compared to 8% of non et al., 2003). Class IV patients treated with bucindolol had a Blacks (FIG. 18). In subjects who were CAR-Del322-325 statistically significant, 1.7 fold increase in the combined 50 carriers, there was a 10% increase in mortality in the bucin endpoint of death or heart failure hospitalization over the first dolol treated cohort, compared to a 29% reduction in mortal 6 months of treatment, whereas Class III patients had no such ity (p=0.031) in subjects who had the wild type CAR (FIG. early adverse effect and overall had a highly significant 19). (p=0.0001), 22% reduction in event rate. The subgroup of Another way to be protected against the sympatholytic patients with the large sympatholytic response to bucindolol 55 effect of bucindolol is to have the high functioning, 389 Arg/ was overrepresented in Class IV patients, as would be Arg variant of the BAR (FIG. 15) (Mason et al., 1999), expected, since this subgroup has very high baseline norepi possessed by 47% of the BEST population. As shown in FIG. nephrine levels (FIG. 15). The subgroup of patients with the 20 and Example 4, whether an individual is 389Arg/Arg (Arg large sympatholytic response to bucindolol also had a worse homozygous) vs. a 389Gly carrier for the BAR is probably a LV and RV function, as would be expected (FIG. 16). 60 major determinant of response to B-agonists, even more so There was a 1.7 fold increase in mortality in BEST in the than the presence or absence of heart failure. It would be subgroup (18% of the treated cohort) that experienced pro expected that individuals who are BAR-389Arg/Arg would found sympatholysis (reduction in norepinephrine by 2244.5 be relatively resistant to the adverse effects of sympatholysis, pg/ml). This subgroup result is similar to that in the MOX since even lower levels of norepinephrine would produce a CONTrial, in patients who received the “pure' sympatholytic 65 relatively robust inotropic response (FIG. 20). That in fact agent moxonidine (Cohn et al., 2003). There appear to be two appears to be the case, as shown in FIG. 19. FIG. 21 indicates pharmacogenetic ways in which patients can be protected that in patients who are CAR-Del322-325 carriers, the pres US 8,080,578 B2 81 82 ence of the BAR-389Arg/Arg receptor converts a 36% Bristow et al., 1998, bucindolol does not increase nighttime increase in mortality in BAR-389Gly carriers to an 18% heart rate on Holter monitoring, considered to be the most mortality reduction. sensitive indicator of ISA in the human heart and a test In addition to protecting against the myocardial depression capable of easily identifying the ISA of Xamoterol or celip resulting from sympatholysis, the BAR-389 Arg/Arg geno ropol (Xameterol Study Group, 1990; Silke et al., 1997). In type confers a greater, “hyper-response' to bucindolol (FIGS. addition, extensive studies performed by the inventors in 5-7). This is likely because the high functioning BAR isolated human heart preparations, bucindolol exhibited no 389Arg/Arg variant confers a greater degree of cardiomyopa evidence of ISA in nonfailing (FIGS. 23 and 24) or failing thy. Because of its higher function, the absolute degree of human hearts (FIGS. 25 and 26). In fact, in failing heart, inhibition of signal transduction by bucindolol is much 10 greater, translating into more potential for benefit in patients carvedilol gives more of a positive inotropic signal than with the BAR-389Arg/Arggenotype. bucindolol, in forskolin pretreated preparations (required to As shown in FIG. 21 and FIG. 20, patients who had the augment signal transduction for detection of weakISA) (FIG. 389Arg/Arg receptor variant had a greater mortality reduc 26). In BEST, the more favorable response of bucindolol in tion response to bucindolol, in the entire cohort (38% reduc 15 high functioning fAR-389 Arg/Arg variant is further evi tion in mortality (p=0.030) vs. a nonsignificant, 10% reduc dence against ISA of bucindolol in human Ventricular myo tion in Gly carriers), and in the CAR wild type patients cardium. (40% reduction in mortality, (p=0.037), vs. a nonsignificant, 22% reduction in Gly carriers). Therefore, the presence or Example 8 absence of the hyper-responder marker BAR-389 Arg/Arg genotype, is the major determinant ofbucindolol response in Best Trial Revisited advanced heart failure patients. FIG. 22 illustrates the pro gression of increasing efficacy for mortality reduction in A DNA substudy of BEST, conducted in 1040 patients, BEST using gene variants to define Subpopulations. For com tested prospective hypotheses regarding two adrenergic parison, the only other f-blocker heart failure mortality trial 25 receptor polymorphisms that, based on work in model sys to enroll a relatively large number (>500) of U.S. patients, tems (Mialet et al., 2003) or in epidemiological heart failure MERIT-HF, is also plotted on FIG. 7. As can be seen, the studies (Small et al., 2002), had the potential to interact with reduction in mortality in BEST increases from annonsignifi the treatment effect of bucindolol. These two adrenergic cant 10% in the entire cohort to 29% in the 80% of patients receptor gene variants, both of which exhibit differential who were CAR wild type, to 38% in the 47% of patients 30 allele frequencies in Blacks vs. non-Black populations, were who were BAR-389Arg/Arg, to 40% in the 40% of patients found to markedly affect treatment outcomes in BEST. The who were both CAR wild type and BAR-389 Arg/Arg. In first was the C. DEL 322-325 polymorphism, a loss of func comparison, the hazard ration for metoprolol CR/XL in U.S. tion gene variant that increases adrenergic drive and predis patients enrolled in MERIT-HF was 1.05 (Wedelet al., 2001). poses to an exaggerated sympatholytic effect of bucindolol. Although it is possible that the BAR-389 genotype-spe 35 Patients who were CDEL322-325 carriers and were treated cific data generated from BEST are generally valid for all with bucindolol had an average reduction in norepinephrine B-blockers, there are good reasons to consider that the find at 3 months of 153-57 (SEM) pg/ml, compared to a reduction ings may apply only to bucindolol. First, as discussed above of 50+13 pg/ml in C. WT/WT patients treated with bucin the AR-389Arg/Arggenotype avoids the adverse effects of dolol (p=0.008). In BEST such exaggerated sympatholytic sympatholysis, and sympatholysis is unique to bucindolol 40 responses were associated with a statistically significant, 1.7 among 3-blockers used to treat heart failure. Second, to maxi fold increase in mortality (Bristow et al., 2004). There were mally inhibit signaling through the high functioning faR two clinical or demographic Subgroups who were predis 389 Arg/Arg receptor, it may actually be useful to decrease posed to marked sympatholysis, Class IV patients and norepinephrine and block the receptor, a combination of Blacks, the only two subgroups who had mortality hazard properties possessed only by bucindolol. Finally, the gene 45 ratios >1.0 in BEST (BEST Writing Committee, 2001). The variant data generated in the BEST trial with bucindolol may allele frequency of C. DEL 322-325 was 0.42 in Blacks, and be considered to be valid only in advanced, Class III-IV heart 0.04 in non-Blacks (p<0.0001). As shown in column 3 of failure. It may well be that in less advanced (NYHA Class Table 9, patients in BEST who were homozygous wild type I-II) heart failure bucindolol would be the drug of choice in (WT/WT) for the C-adrenergic receptor had a 30% reduc subjects with a combination of CAR-Del322-325 carrier 50 tion in all-cause mortality (p=0.031) and a 41% reduction in plus BAR-389 Arg/Arg, since sympatholysis and loss of cardiovascular mortality (p=0.004), whereas patients who myocardial function Support would be less of an issue in this were carriers of the DEL 322-325 polymorphism had a 9% patient population, and individuals who have the haplotype of increase (p-NS) in all-cause mortality, and 3% increase in CAR-Del/Del+fAR-389Arg/Arghave a 10 fold increased cardiovascular mortality (p=NS). Overall, 80% of the BEST risk of developing heart failure (Small et al., 2002). Bucin 55 trial, which was comprised of 24% Blacks, was C. WT/WT, dolol could potentially be the ultimate therapy for these including 34% of Blacks. Thus simply screening for the pres patients, because it lowers norepinephrine (thereby dealing ence of the DEL322-325 polymorphism and treating only the with the impact of the CAR-Del322-325 allele), and blocks 85% of the U.S. population who are homozygous wild type the BAR. for the C2-adrenergic receptor would eliminate the increased There has been controversy in the literature as to whether 60 sympatholysis/mortality risk of bucindolol in advanced heart bucindolol has intrinsic sympathomimetic activity (ISA) in failure populations, and enhance its therapeutic profile. As the human heart, a property that has been offered as the developed below, patients who are O DEL 322-325 carriers explanation for bucindolol's BEST Trial results. Although may be treated with bucindolol if they have the 13,389Arg/ bucindolol clearly has ISA in rodent myocardium, extensive Arg genotype (genotype prevalence in BEST of 0.32 in studies from the inventors indicate that bucindolol has 65 Blacks and 0.51 in non-Blacks), so the population eligible for extremely low inverse agonist activity without any ISA, in bucindolol is 85%+6%=91% of the total U.S. heart failure functioning human Ventricular myocardium. As shown in population, based on racial percentages of 12% Black and US 8,080,578 B2 83 84 88% non-Black. In addition, over 50% of Blacks (34%+ 325 and B 389Gly carriers had a 35% increase in all-cause 21%–55%) could be treated with bucindolol using genotype mortality, and a 36% increase in CV mortality in BEST. The selection. obvious explanation for these adverse responses is that The other adrenergic receptor polymorphism found to advanced heart failure patients who have the low-functioning influence the treatment effect ofbucindolol in BEST was the 389Gly carrier B-adrenergic receptor cannot tolerate the B 389 Arg/Gly SNP, where the Arg/Arg higher functioning exaggerated sympatholytic response associated with the C. variant conferred a “hyper-response' to bucindolol (Table 9, DEL carrier state. In contrast, patients with the high function column 5) compared to the presence of the Gly allele (“Gly ing (389 Arg/Arg) B-receptor, which is characterized by carriers' column 6, Table 9). The evidence that the 389Arg/ robust responses to low concentrations of catecholamines, Arg variant exhibits higher signal transduction function than 10 have no such adverse effect on total or cardiovascular mor Gly carrier variants was provided in the information package tality (Table 9, column 9). Although the relatively small num of the Mar. 28, 2005 meeting, and the evidence that the ber of patients and events precluded statistical significance 389 Argallele is more cardiomyopathic than the 389Gly has for either mortality endpoint in the C. DEL322-325 and B been published by Dr. Liggett’s group (Mialet et al., 2003). As 389Gly carrier subgroup, the congruence of these findings can be seen in Table 9, patients in BEST who were 389Arg/ 15 with both the sympatholytic data and the molecular pharma Arghada 38% (p=0.030) reduction in all-cause mortality, and cology of the adrenergic receptor polymorphisms argues for a 46% reduction in cardiovascular mortality (p=0.015) from their scientific and clinical importance when the issue is bucindolol, compared to respective mortality reductions of patient safety. 10% and 22% (both p=NS) in patients who were 389Gly In BEST, the B 389Arg/Argallele frequency was 0.72 in carriers. Since there is no evidence that carvedilol (Small et non-Blacks, but only 0.57 in Blacks (p<0.0001). Thus in al., 2002) or metoprolol CR/XL (White et al., 2003) possess BEST Blacks had a higher frequency of an allele that predis a markedly differentiated therapeutic response between B posed to increased mortality (C. DEL 322-325), and a lower 389Arg/Arg and B 389Gly carriers, it is likely that bucin frequency of the “hyper-response B 389Arg/Arg allele. dolol's salutary effects in patients with the B 389 Arg/Arg Both of these genetic differences in American Blacks likely receptor gene variant are due to the combination of lowering 25 contributed to the trend towards an adverse outcome in this norepinephrine signaling and receptor competitive antago demographic group (BEST Writing Committee, 2001). nism. In that regard, Dr. Bristow's group has presented evi The pharmacogenomic substudies of BEST used prospec dence based on physiologic and molecular/bio marker data tive hypotheses based on the anticipated pharmacologic inter that heart failure patients treated withfull therapeutic doses of action of bucindolol with specific adrenergic receptor poly B-blocking agents exhibit evidence of ongoing myocardial 30 morphisms that had been previously extensively investigated adrenergic signaling (Lowes et al., 2001), and so lowering of in model and human systems. Accordingly, it is believed that norepinephrine in patients with the B 389Arg/Arghigh func these pharmacogenomic hypotheses are more valid than stan tioning receptor variant may offer additional benefit. dard, retrospectively derived, Subgroup analyses. Moreover, patients who were both B 389Arg/Arg and C The pharmacogenetic data analyzed from the BEST trial WT/WT (column 8, Table 9) had a 40% reduction in all-cause 35 are included below. The population that agreed to the DNA mortality (p=0.037), a 47% reduction in cardiovascular mor Substudy and pharmacogenomic analysis was not different tality (p=0.020), and a 39% reduction in mortality+heart fail from the entire cohort in baseline characteristics. In addition, ure hospitalization (p=0.002), or therapeutic responses that there was no evidence of a gene dose effect for either poly are much greater than in the entire BEST cohort or in the morphism; heterozygote effects were the same or greateras in cognate opposite diplotypes. In that regard, column 11 in 40 homozygotes. Because of this, both the CDEL322-325 and Table 9 indicates that patients who were both C. DEL 322 B 389Gly alleles are assumed to act as dominant negatives. TABLE 9
BEST BEST BEST BEST C. DEL BEST B 389 Gly entire cohort Clawtwit carrier f 389Arg/Arg carrier bucindolol bucindolol bucindolol bucindolol bucindolol (n = 2708) (n = 829/1036) (n=207/1036) (n = 493/1040) (n = 547/1040) mean fu mean fu mean fu mean fu mean fu Endpoint 2.0 yrs 2.0 yrs 2.0 yrs 2.0 yrs 2.0 yrs Mortality 0.90; 860 Ev 0.70; 155 Ev 1.09:37 Ev 0.62; 82 Ev 0.90: 111 Ev (0.78, 1.02) (0.51, 0.97) (0.57, 2.08) (0.40, 0.96) (0.62, 1.30) b = 0.10 p = 0.031 p = 0.79 p = 0.030 b = 0.57 CV Mortality 0.86; 731 Ev 0.59; 130 Ev 1.03: 32 Ev 0.54; 66 Ev 0.78; 97 Ev (0.74, 0.99) (0.42, 0.85) (0.52, 2.07) (0.33, 0.89) (0.52, 1.18) b = 0.040 p = 0.004 p = 0.92 p = 0.015 b = 0.24 Mortality + HF 0.81: 1421 Ev 0.72: 341 Ev 0.89: 84 Ev 0.66; 190 Ev 0.87: 236 Ev Hospitalization (0.73, 0.90) (0.59, 0.90) (0.58, 1.37) (0.50, 0.88) (0.62, 1.30) < O.OOO1 p = 0.003 p = 0.60 p = 0.004 b = 0.25 HF 0.78: 1045 Ev 0.74; 267 Ev 0.76; 67 Ev 0.64; 154 0.86; 181 Ev Hospitalization (0.69, 0.88); (0.58, 0.95) (0.47, 1.24) (0.48, 0.90) (0.64, 1.15) < 0.001 p = 0.016 p = 0.27 p = 0.006 b = 0.30
BEST BEST BEST MERITHF BEST B 389 Arg/Arg-- B 389 Gly B 389 Gly US Pts B 389Arg, Arg + C. DEL carriers + C2 carriers + DEL metoprolol oWTWT carriers WTWT carriers CRXL bucindolol bucindolol bucindolol bucindolol (n = (n = 418/1036) (n = 73/1036) (n = 411/1036) (n = 134/1036) 1071/3991) mean ful 2 O mean ful 2.0 mean ful 2.0 mean ful 2.0 mean fu 1.0 US 8,080,578 B2 85 86 TABLE 9-continued Endpoint yrs yrs yrs yrs yrs Mortality 0.60; 69 Ev 0.71; 13 Ev 0.82; 86 Ev 1.35; 24 Ev 1.05; 100 Ev (0.38, 0.97) (0.24, 2.11) (0.54, 1.26) (0.61, 3.02) 0.71, 1.56 p = 0.037 p = 0.53 p = 0.37 p = 0.46 p = NS CV Mortality 0.53: 56 Ev 0.58; 73 Ev 0.67; 411 Ev 1.36; 22 Ev -0.96:90 Ev (0.31, 0.90) (0.17, 2.02) (0.42, 1.07) (0.59.3.15) p = NS p = 0.020 p = 0.39 p = 0.09 p = 0.47 Mortality + HF 0.61: 156 Ev 0.85; 34 Ev 0.86; 185 Ev 0.81: 50 Ev -0.84; 200 Ev Hospitalization (0.44, 0.84) (0.43, 1.69) (0.64, 1.16) (0.46, 1.43) (0.61, 1.12) p = 0.002 p = 0.64 p = 0.32 p = 0.47 p = NS HF 0.59; 126 Ev 0.81: 73 Ev 0.93; 411 Ev 0.62; 134 Ev NA Hospitalization (.41, .. 84) (0.38, 1.72 (0.67, 1.29) (0.32, 1.21) p = 0.004 p = 0.59 p = 0.66 p = 0.16 Effects of B-blockers vs. placebo in the only available published data from intention-to-treat mortality trials conducted in U.S. heart failure patients, Ev = Events;NA, not available
Example 9 bucindolol would have a slight advantage compared to meto prolol CR/XL in the remaining genotypes being treated in a Additional Studies: Design I pure Chomozygous wild type (WT/WT) adrenergic recep tor background. For ethical reasons it is no longer possible to perform The primary endpoint of the trial is noninferiority against placebo-controlled trials with B-blockers in patients with metoprolol CR/XL (TOPROL-XL), using the 95% upper heart failure caused by a primary or secondary dilated cardi 25 confidence limit (UCL) of the time to mortality plus heart omyopathy. This leaves as design options non-inferiority or failure hospitalization endpoint hazard ratio that was mea Superiority studies against an active B-blocker control, or sured in the MERIT-HF trial (Hjalmarson et al. 2000). alternatively comparison of the bucindolol response between Though the MERIT-HF Trial included all genotypes, only gene variants. A non-inferiority design option would appear 208 (5.2%) of the 3991 enrolled patients were Black. Alpha to be eliminated by the lack of receptor gene variant data for 30 322-325 DEL carriers are highly over-represented in Black any other agent (among other Phase III trials only MERIT-HF subjects (in the BEST Trial the C-322-325 DEL allele fre had a pharmacogenomic substudy (Small et al., 2002), and quency was 0.423 in Blacks vs. 0.043 in non-Blacks, while that was too small to reach any meaningful conclusions). the DEL carrier prevalence was 66% in Blacks, vs. 8% in Shown in FIG. 27 as Study Design I is a possible study non-Blacks). Therefore, the MERIT-HF Trial was likely scheme that would compare the combination of genotype 35 approximately 90% C wild type, or comparable genotypi targeting and bucindolol to non-targeted therapy with meto cally to the proposed study population in Design II. In addi prolol CR/XL, using a primary endpoint of time to death or tion, since metoprolol CR/XL has no sympatholytic effects heart failure hospitalization. In Design I, patients randomized there is no reason to expect a differential response to meto to bucindolol who are BAR-389Gly carriers would not be prolol CR/XL in C wild type vs. DEL carriers. The 95% entered into the study, since based on BEST data the response 40 upper confidence limit for the hazard ratio (UCL) for bucin to bucindolol is not sufficient to warrant further evaluation. dolol:metoprolol CR/XL (“noninferiority margin') has been These patients would instead enter a registry where a mini determined from the formula (Hasselbladet al., 2001) (UCL mum of information, likely confined to vital status, would be bucindolol:metoprolol CR/XL)*(UCL metoprolol CR/XL: captured as they are treated in whatever way their treating placebo)s 1.0}; using the MERIT-HF entire cohort UCL of physicians choose. The primary comparison of Design I 45 0.80 for metoprolol: placebo yields x*0.80s 1.0, or a nonin would be between BAR-389Arg/Arg patients treated with feriority margin Xs 1.25. The 1.25 value was then reduced to bucindolol, and all genotypes treated with metoprolol CR/XL 1.16, to provide greater certainty on noninferiority. The target (TOPROL XL) non-targeted therapy. The total randomized UCL of 1.16 is also the value obtained when scaling the sample size for Design I would be 900, with 662 subjects MERIT-HF hazard ratio/UCL up from the observed values of participating in the primary outcome comparison. 50 0.69/0.80 to a hazard ratio of 1.0. The power estimate of 85% for a 2-sided C-s0.05 was then determined from an expected Example 10 hazard ratio of 0.90 (the slight advantage of bucindolol in an C. WT/WT population, gained through bucindolol's better Additional Studies: Design II inhibition of B-389 Arg/Arg and/or its myocardial B-recep 55 tor blockade). In Study Design II (FIG.28) for an additional study design, Support for an advantage of bucindolol over metoprolol patients with symptomatic and advanced heart failure (rein CR/XL is provided by the U.S. enrolled patients in MERIT forced by the heart failure hospitalization history over the HF (Table 9), who when treated with metoprolol CR/XL had preceding year, therefore, patients who are Class II at the time a mortality increase of 5% and a mortality+HF hospitaliza of screening are allowed in the trial) and LVEF's s().35 (the 60 tion decrease of 16%. In contrast, in BEST the entire cohort general description of the BEST Trial population) are initially had a mortality reduction of 10% (p=0.10) and a mortality+ screened for the absence of the C-322-325 DEL polymor HF hospitalization decrease of 19% (p<0.0001), while the phism, in the carrier state (either heterozygous or homozy C. wild type patients had a mortality reduction of 30% gous). This restriction eliminates much of the adverse effect (p=0.031), and a mortality plus HF hospitalization reduction of sympatholysis from bucindolol, primarily manifested as a 65 of 28% (p=0.003). Dividing the hazard ratios (0.72/0.84) trend for increased mortality in BEST (hazard ratio 1.09, yields an expected hazard ratio of 0.86, and an effect size for column 4, Table 9) and in MOXCON, and it is anticipated that bucindolol vs. metoprolol CR/XL of 1.00-0.86, or 0.14. Thus US 8,080,578 B2 87 88 it is not unreasonable to expect a 10% difference in favor of CR/XL over placebo, is deemed insufficient for demonstrat bucindolol vs. metoprolol CR/XL in a U.S. population that is ing efficacy, another design is proposed. An UCL of 1.14. CWT/WT. By the criterion proposed in FIG. 28, regardless which would preserve 50% of the metoprolol CR/XL vs. of the bucindolol/metoprolol hazard ratio, if the UCL is placebo treatment effect at 90% power, was considered <1.16, the conclusion would be that bucindolol is noninferior acceptable. Therefore, the design shown in FIG. 28 has been to metoprolol CR/XL for effects on time to mortality or HF hospitalization, in an advanced HF population that is 100% adjusted to achieve this goal, with a power of 90% (FIG. 29). C-adrenergic receptor homozygous wild type. The increase in statistical power required to lower the UCL The second part of the trial design is intended to provide has been accomplished by increasing the sample size from additional evidence that pharmacogenomically targeted 1300 to 1600, and to a lesser extent by converting from a 2:1 bucindolol is superior to non-targeted metoprolol CR/XL, as 10 to a 1:1 randomization between bucindolol and metoprolol a secondary endpoint. Here the population treated with bucin CR/XL. Also, in response to agency feedback we have added dolol is 3-389 Arg/Arg patients who also are C wild type. another secondary endpoint, bucindolol vs. metoprolol In BEST 47% of the entire cohort was B-389 Arg/Arg, as CR/XL in B-389 Arg/Arg patients. The power estimate for were 50% of the C wild type patients. As shown in Table 9, this secondary endpoint, based on an expected effect size of the diplotype of B-389 Arg/Arg--O wild type exhibited a 15 25%, is 71%, while the power estimate for the other second 40% reduction in mortality (p=0.037), a 39% reduction in ary endpoint, based on an expected effect size of 27%, is 88%. mortality+HF hospitalization (p=0.002), and a 41% reduc The former effect size of 25% was difficult to estimate tion in HF hospitalization (p=0.004). The expected hazard because of limited data with metoprolol CR/XL in a B-389 ration for mortality+HF hospitalizations for bucindolol:me Arg/Arg population; the only data available Suggest little or toprolol CR/XL (MERIT U.S. data) would be 0.61/0.84, or no enhancement of the metoprolol CR/XL treatment effect 0.73. Using an effect size of 27% in bucindolol-treated compared to placebo (White et al., 2003). The effect size of B-389 Arg/Arg--C wild type diplotype patients vs. meto 27% for the other secondary endpoint is based on U.S. prolol CR/XL yields a power calculation of 81%. Thus in the MERIT-HF data as discussed above. proposed trial shown in FIG. 28, the noninferiority margin for The foregoing description is considered as illustrative only the primary endpoint is conservatively based on the entire 25 cohort results of MERITHF, while the power calculations for of the principles of the invention. Further, since numerous both the primary and secondary endpoints incorporate an modifications and changes will readily occur to those skilled expected advantage of bucindolol in the selected genotypes, in the art, it is not desired to limit the invention to the exact based on actual data in U.S. populations for both bucindolol construction and process as described above. Accordingly, all and metoprolol CR/XL. Support for the validity of this com Suitable modifications and equivalents may be resorted to parison is that in the BEST Trial DNA substudy, B-389 30 falling within the scope of the invention as defined by the Arg/Arg patients treated with placebo had identical event claims that follow. The words “comprise.” “comprising.” rates to B-389 Gly carrier patients treated with placebo “include.” “including,” and “includes” when used in this (FIGS. 6 and 7). In other words, as shown in FIG. 7, the more specification and in the following claims are intended to favorable event rate in B-389 Arg/Arg patients treated with specify the presence of stated features, integers, components, bucindolol is entirely due to the treatment effect, not to a 35 or steps, but they do not preclude the presence or addition of better natural history of patients with the f-389 Arg/Arg one or more other features, integers, components, steps, or genotype. This 1st order secondary endpoint will provide groups thereof. clinicians with further evidence that genotype targeting with bucindolol produces better clinical results than non-targeted REFERENCES therapy with an approved heart failure f3-blocker. 40 In the trial design shown in FIG. 28, the population treated The following references, to the extent that they provide with bucindolol is B-389 Arg/Arg patients who also are C. exemplary procedural or other details Supplementary to those wild type. In BEST 47% of the entire cohort was B-389 set forth herein, are specifically incorporated herein by refer Arg/Arg, as were 50% of the C wildtype patients. As shown CCC. in Table 9, the diplotype of B-389 Arg/Arg--C wild type 45 U.S. Pat. No. 3,817,837 exhibited a 40% reduction in mortality (p=0.037), a 39% U.S. Pat. No. 3,850,752 reduction in mortality plus HF hospitalization (p=0.002), and U.S. Pat. No. 3,939,350 a 41% reduction in HF hospitalization (p=0.004). The U.S. Pat. No. 3,996,345 expected hazard ration for mortality plus HF hospitalizations U.S. Pat. No. 4,196,265 for bucindolol:metoprolol CR/XL (MERIT U.S. data) would 50 U.S. Pat. No. 4,275, 149 be 0.61/0.84, or 0.73. Using an effect size of 27% in bucin U.S. Pat. No. 4,277,437 dolol-treated f-389 Arg/Arg plus C wild type diplotype U.S. Pat. No. 4,366,241 patients vs. metoprolol CR/XL yields a power calculation of U.S. Pat. No. 4,582,788 81%. Thus in the proposed trial shown in FIG. 28, the non U.S. Pat. No. 4,627,429 inferiority margin for the primary endpoint is conservatively 55 U.S. Pat. No. 4,659,774 based on the entire cohort results of MERITHF, while the U.S. Pat. No. 4,683,194 power calculations for both the primary and secondary end U.S. Pat. No. 4,683,195 points incorporate an expected advantage ofbucindolol in the U.S. Pat. No. 4,683,202 selected genotypes, based on actual data in U.S. populations U.S. Pat. No. 4,683,202 for both bucindolol and metoprolol CR/XL. 60 U.S. Pat. No. 4,683,202 U.S. Pat. No. 4,784,857 Example 11 U.S. Pat. No. 4,800,159 U.S. Pat. No. 4,816,571 Additional Studies: Design III U.S. Pat. No. 4,883,750 65 U.S. Pat. No. 4,946,773 In case the noninferiority margin of 1.16, which provides a U.S. Pat. No. 4,959,463 36% preservation of the treatment effect of metoprolol U.S. Pat. No. 4,965,188 US 8,080,578 B2 89 90 Pat. 5,126,145 U.S. Pat. No. 5,935,791 Pat. 5,130.238 U.S. Pat. No. 5,935,825 Pat. 5,141,813 U.S. Pat. No. 5,939,291 Pat. 5,169,766 U.S. Pat. No. 5,942,391 Pat. 5,264,566 U.S. Pat. No. 5,952,174 Pat. 5,279,721 U.S. Pat. No. 6,113,940 Pat. . 5,428,148 U.S. Pat. No. 4,656,127 Pat. 5,554,744 U.S. Pat. No. 4,682,195 Pat. 5,574,146 Abbondanzo et al., Breast Cancer Res. Treat., 16:182(151), Pat. . 5,602,244 10 1990. Pat. 5,605,798 Allred et al., Arch. Surg., 125(1): 107-113, 1990. Pat. 5,645,897 Anand et al., Circulation, 107(9): 1278-1283, 2003. Pat. 5,662,925 Anderson et al., J. Card. Fail., 9:266-277, 2003. Pat. 5,705,629 Asano et al., J. Cardiovasc. Pharmacol., 37:678-691, 2001. Pat. 5,788,983 15 Ausubel et al., In. Current Protocols in Molecular Biology, Pat. 5,840,873 John, Wiley & Sons, Inc, New York, 1989. Pat. 5,843,640 Barany, et al., Proc. Natl. Acad. Sci. USA, 88:189-193, 1991 Pat. 5,843,650 Bellus, J. Macromol. Sci. Pure Appl. Chem. A31(1): 1355 Pat. 5,843,651 1376, 1994. Pat. 5,843,663 Benedictet al., Circulation, 94(4):690-697, 1996. Pat. 5,846,708 BEST Trial Investigators, N. Engl. J. Med., 344(22): 1659 Pat. 5,846,709 1667, 2001. Pat. 5,846,717 Bolger, Int J Cardiol, 92(1):1-8, 2003. Pat. 5,846,726 Bouzamondo et al., Fund. Clin. Pharmacol., 15:95-109, Pat. 5,846,729 25 2001. Pat. 5,846,783 Bristow etal, Circ. Res., 59(3):297-309, 1986. Pat. 5,849,481 Bristow et al., Cardiovasc/Drugs. Then, 11:291-296, 1997. Pat. 5,849,483 Bristow et al., Circulation, 110:1437-1442, 2004. Pat. 5,849,486 Bristow et al., Circulation, 89(4):1632-1642, 1994. Pat. 5,849,487 30 Bristow et al., Clin. Cardiol., 21 (12 Suppl 1): 13-13, 1998. Pat. 5,849,497 Bristow et al., J Card Fail., 9(6):444-53, 2003. Pat. 5,849,546 Bristow et al., Mol. Pharmacol., 35:296-303, 1988. Pat. 5,849,547 Bristow, Circulation, 101:558-569, 2000. Pat. 5,851,770 Brodde et al., J. Cardiovasc., Pharmacol., 8:1235-1242, Pat. 5,851,772 35 1986. Pat. 5,853,990 Brodde et al., Z. Kardiol., 81:71-78, 1992. Pat. 5,853,992 Brown et al., Immunol Ser, 53:69-82, 1990. Pat. 5,853,993 Brum et al. Am. J. Physiol. Heart Circ. Physiol., 283:H1838 Pat. 5,856,092 18345, 2002. Pat. 5,858,652 40 Capaldi et al., Biochem. Biophys. Res. Comm., 74(2):425 Pat. . 5,861,244 433, 1977. Pat. 5,863,732 CIBIS-II Investigators, Lancet, 353:9-13, 1999. Pat. 5,863,753 Cohn et al., Eur: J. Heart Fail., 5:659-667, 2003. Pat. 5,866,331 Cohn et al., N. Engl. J. Med., 311:819-823, 1984. Pat. 5,866,337 45 de Arruda et al., Expert Rev. Mol. Diagn., 205):487-496, 2002. Pat. 5,866,366 De Jager et al., Semin. Nucl. Med., 23(2):165-179, 1993. Pat. 5,900,481 Dohlman et al., Annu. Rev. Biochem., 60:653-688, 1991. Pat. 5,905,024 Domanski et al., J. Am. Coll. Cardiol., 42:914-922, 2003. Pat. 5,910.407 Doolittle et al., Methods Mol. Biol., 109:215-237, 1999. Pat. 5,912,124 50 Eichhornet al., Am. J. Cardiol. 79:794-798, 1997. Pat. 5,912,145 Epstein et al., Ann. Inn. Med., 65:20-7, 1968. Pat. 5,912,148 European Applin. 201,184 Pat. 5,916,776 European Applin. 237,362 Pat. 5,916,779, European Applin. 258,017 Pat. 5,919,626 55 European Applin. 266.032 Pat. 5,919,630 European Applin. 320308 Pat. 5,922,574 European Applin. 329,822 Pat. 5,925,517 European Applin. 50,424 Pat. 5,925,525 European Applin. 84,796 Pat. 5,928,862 60 Fowler and Bristow, Am. J. Cardiol., 55(10)D120-D124. Pat. 5,928,869 1985. Pat. 5,928,870 Francis et al., Circulation, 87(6 Suppl):VI40-8, 1993. Pat. 5,928,905 French Applin. 2,650,840 Pat. 5,928,906 Frielleet al., Proc. Natl. Acad. Sci. USA, 84.7920-7924, 1987. Pat. 5,929,227 65 Froehler et al., Nucleic Acids Res., 14(13):5399-5407, 1986. Pat. 5,932,413 Frohman, In: PCR Protocols. A Guide To Methods And Appli Pat. 5,932,451 cations, Academic Press, N.Y., 1990. US 8,080,578 B2 91 92 Gaffney et al., Circ Res., 12:264-268, 1963. Ohara et al., Proc. Natl. Acad. Sci. USA, 86:5673-5677, 1989. Gilbert et al., Amer. J. Med., 88:223-229, 1990. Orita et al., Genomics, 5:874-879, 1989. Granger et al., Lancet., 362:772-776, 2003. Packer et al., Circulation, 106(17):2194-2199, 2002. Great Britain Applin 2 202328 Packer et al., N. Engl. J. Med., 344:1651-1658, 2001. Green et al., 1994 PCT Appln. PCT/US87/00880 Gulbis and Galand, Hum. Pathol., 24(12): 1271-1285, 1993. PCT Appln. PCT/US89/01025 PCT Appln. WO 88/10315 Halushka et al., Nat. Genet., 22(3):239-247,1999. PCT Appln. WO 89/06700 Harlow and Lane. In: Antibodies. A Laboratory Manual, PCT Applin. WO93/224.56 Cold Spring Harbor Laboratory, 1988 PCT Appln. WO95/11995 Hasselbladet al., Drug Information.J., 35:435-449, 2001. 10 PCT Appln. WO89/06700 Hein et al., Nature, 402:181-184, 1999. PCT Applin. WO90/01069 Hershberger et al., J. Cardiovasc. Pharm., 15:959-967, 1990. PCT Appln. WO91/02087 Hjalmarson et al., J. Am. Med. Assoc., 283: 1295-1302, 2000. PCT Appln. WO92/15712 Humphries, et al., In: Molecular Diagnosis of Genetic Dis Perez et al., Nature Med., 9:1300-1305, 2003. eases, Elles (Ed.), 321-340, 1996. 15 Physicians Desk Reference' Innis et al., Proc. Natl. Acad. Sci. USA, 85 (24):9436-9440, Pollock et al., Am. J. Cardiol. 66:603-607, 1990. 1988. Port et al., Mol. Pharmacol., 60(4):629-631, 2001. Isnard et al., Am. J. Cardiol., 86(4):417-421, 2000. Prezant et al., Hum. Mutat., 1:159-164, 1992. Johnson et al., Clin. Pharmacol. Ther, 73:366-71, 2003. RathZ. et al., J. Cardiovasc. Pharmacol., 39:155-160, 2002. Johnson et al., Clin. Pharmacol. Ther, 74(1):44-52, 2003. Remington’s Pharmaceutical Sciences' 15th Edition, pages Jones, Nature, 199:280-282, 1963. 1035-1038 and 1570-1580 Joseph et al., Br. J. Pharm. 142:51-56, 2004. Rockman et al., Am. J. Cardiol., 64(19): 1344-1348, 1989. Kalbfleisch et al., 1980 Ruano et al., Nucl. Acids Res., 19:6877-6882, 1991. Kaplan et al., JAmer: Statistical Assoc., 53:457-481, 1958. Ruano et al., Nucl. Acids Res., 17:8392, 1989. Kaye et al., J. Am. Coll. Cardiol., 26(5):1257-1263 1995. 25 Sallach et al., Ann. Med., 35:259-266, 2003 Ke and Cardon Bioinformatics, 19(2):287-288, 2003. Salomon, Methods Enzymol., 195:22-28, 1991. Klaassen, In: The Pharmacological Basis of Therapeutics, Sambrook et al., In: Molecular cloning, Cold Spring Harbor Goodman and Gilman, Eds. Pergamon Press, 8th Ed., pp. Laboratory Press, Cold Spring Harbor, N.Y., 2001. 49-61, 1990. Sanger et al., J. Molec. Biol., 94:441, 1975. Krum et al. Circulation, 92: 1499-1506, 1995. 30 SAS (r) proprietary software, release 6.12. Cary, N.C.: SAS Kuppuswamy, et al., Proc. Natl. Acad. Sci. USA, 88:1143 Institute, 1996 1147, 1991. Sederberg et al., J. Am. Coll. Cardiol. 35(2) 207A:147, 2000. Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173, 1989. Sheffield, et al., Proc. Natl. Acad. Sci. USA, 86:232-236, Kwok and Chen, Curr Issues Mol. Biol., April 5(2):43-60. 1989. 2003. 35 Silke et al., J. Cardiovasc. Pharmacol., 30(6):817-823, 1997. Kwok et al., Genomics, 23(1): 138-144, 1994. Small et al. J. Biol. Chem., 275:23059-23064, 2000. Kwok, Annu. Rev. Genomics Hum. Genet., 2:235-258, 2001. Small et al., N. Engl. J. Med., 347: 1135-1142, 2002. Landegren, et al., Science 241:1077-1080, 1988. Sofowora et al., Clin Pharmacol Then, 73:366-71, 2003. Landegren, et al., Science, 241:1077-1080, 1988. Sokolov, Nucl. Acids Res. 18:3671, 1990. Levin et al., J Biol. Chem., 277(34):30429-30435, 2002. 40 Stephen, Am. J. Cardiol., 18:463-472, 1966. Liggett et al., In: Catecholamines, Bouloux (Ed.). W. B. Stevens et al., Biotechniques, 34:198-203, 2003. Sounders, London, 1993. Strosberg, Annu. Rev. Pharmacol. Toxicol., 37:421-450, Liggett, J. Clin. Invest., 107:947-948, 2001. 1997. Liu et al., Clin. Pharmacol. Ther, 74:372-9, 2003. Swedberg et al., Circulation, 105(15):1797-803, 2002. Lohse, Trends Mol. Med., 10:55-58, 2004. 45 Swedberg et al., Circulation, 82(5):1730-1736, 1990. Lowes et al., Circulation, 102:11-628, 2000. Swedberg et al., Eur: Heart J., 17(9): 1306-1311, 1996. Lowes et al., Circulation, 104:11-436, 2001. Taillon-Miller et al., Genome Res, 8(7):748-754, 1998. Lowes et al., N. Engl. J. Med., 346:1357-1365, 2002. Taylor et al., Cong. Heart Failure, 10:281-288, 2004. Mann et al., Circulation, 111(21):2837-2849, 2005. The Merck Index, Eleventh Edition Mason et al., J. Biol. Chem., 274:12670-12674, 1999. 50 Trial Investigators, 2001 Maxam, et al., Proc. Natl. Acad. Sci. USA, 74:560, 1977. Turki, et al., J. Clin. Invest., 95:1635-1641, 1995. McMurray et al., Lancet., 362:767-771, 2003. Ugozzollet al., GATA 9:107-112, 1992. MERIT-HFStudy Group, Lancet., 353:2001-2007, 1999. Van Campen et al., J. Cardiovasc. Pharmacol., 32 Suppl Meyers, et al., Science, 230:1242, 1985. 1:S31-S35, 1998. Mialet et al., Nat. Med., 9:1300-1305, 2003. 55 Waagstein et al., Lancet., 342:1441-1446, 1993. Modrich, Ann. Rev. Genet., 25:229-253, 1991. Wagoner et al., Am. Heart J., 144(5):840-846, 2002. Mullis et al., Cold Spring Harbor Symp. Ouant. Biol. 51:263 Walker, et al., Proc. Natl. Acad. Sci. USA, 89:392–396, 1992. 273, 1986. Wartell, et al., Nucl. Acids Res., 18:2699-2706, 1990. Nakamura et al., In: Handbook of Experimental Immunology Wedel et al., Am. Heart J., 142:502-511, 2001. (4"Ed.), Weir et al., (eds). 1:27, Blackwell Scientific Publ., 60 White et al., Eur: J. Heart Fail., 5:463-468, 2003. Oxford, 1987. Winter, et al., Proc. Natl. Acad. Sci. USA, 82:7575, 1985. Neumister et al., Pharmacogenet. Genomics, 15:143-149, Xameterol Study Group, 1990 2005. Yancy et al., J. Am. Coll. Cardiol., 29:284A, 1997. Nickerson et al., Proc. Natl. Acad. Sci. USA, 87:8923-8927, Yancy et al., N. Engl. J. Med., 344(18): 1358-1365, 2001. 1990. 65 Yang-Feng et al., Proc. Natl. Acad. Sci. USA, 87: 1516-1520, Nyrenet al., Anal. Biochem. 208:171-175, 1993. 1990. O'Shaughnessy et al., Clin. Sci., 99:233-238, 2000. Zhu et al., Proc. Natl. Acad. Sci. USA, 98(4):1607-12, 2001.
US 8,080,578 B2 97 98 - Continued
Lell Wall Pro Ala Ser Pro Pro Ala Ser Luell Luell Pro Pro Ala Ser Glu 35 4 O 45
Ser Pro Glu Pro Lell Ser Glin Glin Trp Thir Ala Gly Met Gly Luell Luell SO 55 60
Met Ala Luell Ile Wall Lell Lell Ile Wall Ala Gly Asn Wall Luell Wall Ile 65 70
Wall Ala Ile Ala Lys Thir Pro Arg Luell Glin Thir Lell Thir Asn Luell Phe 85 90 95
Ile Met Ser Luell Ala Ser Ala Asp Luell Wall Met Gly Lell Luell Wall Wall 105 11 O
Pro Phe Gly Ala Thir Ile Wall Wall Trp Gly Arg Trp Glu Tyr Gly Ser 115 12 O 125
Phe Phe Glu Lell Trp Thir Ser Wall Asp Wall Lell Wall Thir Ala 13 O 135 14 O
Ser Ile Glu Thir Lell Cys Wall Ile Ala Luell Asp Arg Luell Ala Ile 145 150 155 160
Thir Ser Pro Phe Arg Glin Ser Luell Luell Thir Arg Ala Arg Ala Arg 1.65 17O
Gly Luell Wall Cys Thir Wall Trp Ala Ile Ser Ala Lell Wall Ser Phe Luell 185 19 O
Pro Ile Luell Met His Trp Trp Arg Ala Glu Ser Asp Glu Ala Arg Arg 195
Tyr Asn Asp Pro Cys Asp Phe Wall Thir Asn Arg Ala Tyr 21 O 215
Ala Ile Ala Ser Ser Wall Wall Ser Phe Tyr Wall Pro Lel Ile Met 225 23 O 235 24 O
Ala Phe Wall Lell Arg Wall Phe Arg Glu Ala Glin Glin Wall Lys 245 250 255
Ile Asp Ser Glu Arg Arg Phe Luell Gly Gly Ala Arg Pro 26 O 265 27 O
Pro Ser Pro Ser Pro Ser Pro Wall Pro Ala Pro Ala Pro Pro Gly
Pro Pro Arg Pro Ala Ala Ala Ala Ala Thir Ala Pro Lel Ala Asn Gly 29 O 295 3 OO
Arg Ala Gly Arg Pro Ser Arg Luell Wall Ala Lel Arg Glu Glin 3. OS 310 315
Ala Luell Thir Lell Gly Ile Ile Met Gly Wall Phe Thir Luell 3.25 330 335
Trp Luell Pro Phe Phe Lell Ala Asn Wall Wall Ala Phe His Arg Glu 34 O 345 35. O
Lell Wall Pro Asp Arg Lell Phe Wall Phe Phe ASn Trp Lel Gly Tyr Ala 355 360 365
Asn Ser Ala Phe Asn Pro Ile Ile Arg Ser Asp Phe Arg 37 O 375
Lys Ala Phe Glin Gly Lell Lell Ala Arg Arg Ala Ala Arg Arg 385 390 395 4 OO
Arg His Ala Thir His Gly Asp Arg Pro Arg Ala Ser Gly Luell Ala 4 OS 41O 415
Arg Pro Gly Pro Pro Pro Ser Pro Gly Ala Ala Ser Asp Asp Asp Asp 425 43 O
Asp Asp Wall Wall Gly Ala Thir Pro Pro Ala Arg Lell Lell Glu Pro Trp 435 44 O 445
Ala Gly Asn Gly Gly Ala Ala Ala Asp Ser Asp Ser Ser Luell Asp
US 8,080,578 B2 103 104 - Continued
85 90 95
Ile Met Ser Luell Ala Ser Ala Asp Luell Wall Met Gly Lell Luell Wall Wall 105 11 O
Pro Phe Gly Ala Thir Ile Wall Wall Trp Gly Arg Trp Glu Gly Ser 115 12 O 125
Phe Phe Glu Lell Trp Thir Ser Wall Asp Wall Lell Wall Thir Ala 13 O 135 14 O
Ser Ile Glu Thir Lell Cys Wall Ile Ala Luell Asp Arg Luell Ala Ile 145 150 155 160
Thir Ser Pro Phe Arg Glin Ser Luell Luell Thir Arg Ala Arg Ala Arg 1.65 17O
Gly Luell Wall Cys Thir Wall Trp Ala Ile Ser Ala Lell Wall Ser Phe Luell 185 19 O
Pro Ile Luell Met His Trp Trp Arg Ala Glu Ser Asp Glu Ala Arg Arg 195
Tyr Asn Asp Pro Cys Asp Phe Wall Thir Asn Arg Ala 21 O 215
Ala Ile Ala Ser Ser Wall Wall Ser Phe Wall Pro Lel Ile Met 225 23 O 235 24 O
Ala Phe Wall Lell Arg Wall Phe Arg Glu Ala Glin Glin Wall 245 250 255
Ile Asp Ser Cys Glu Arg Arg Phe Luell Gly Gly Ala Arg Pro 26 O 265 27 O
Pro Ser Pro Ser Pro Ser Pro Wall Pro Ala Pro Ala Pro Pro Gly
Pro Pro Arg Pro Ala Ala Ala Ala Ala Thir Ala Pro Lel Ala Asn Gly 29 O 295 3 OO
Arg Ala Gly Arg Pro Ser Arg Luell Wall Ala Lel Arg Glu Glin 3. OS 310 315
Ala Luell Thir Lell Gly Ile Ile Met Gly Wall Phe Thir Luell 3.25 330 335
Trp Luell Pro Phe Phe Lell Ala Asn Wall Wall Ala Phe His Arg Glu 34 O 345 35. O
Lell Wall Pro Asp Arg Lell Phe Wall Phe Phe ASn Trp Lel Gly Ala 355 360 365
Asn Ser Ala Phe Asn Pro Ile Ile Arg Ser Asp Phe Arg 37 O 375
Lys Ala Phe Glin Arg Lell Lell Ala Arg Arg Ala Ala Arg Arg 385 390 395 4 OO
Arg His Ala Thir His Gly Asp Arg Pro Arg Ala Ser Gly Luell Ala 4 OS 415
Arg Pro Gly Pro Pro Pro Ser Pro Gly Ala Ala Ser Asp Asp Asp Asp 425 43 O
Asp Asp Wall Wall Gly Ala Thir Pro Pro Ala Arg Lell Lell Glu Pro Trp 435 44 O 445
Ala Gly Asn Gly Gly Ala Ala Ala Asp Ser Asp Ser Ser Luell Asp 450 45.5 460
Glu Pro Arg Pro Gly Phe Ala Ser Glu Ser Wall 465 470 47s
SEO ID NO 5 LENGTH: 1958 TYPE: DNA ORGANISM: Homo sapiens FEATURE:
US 8,080,578 B2 109 110 - Continued
Gly Ala Wall Ala Gly Lell Ala Ala Wall Wall Gly Phe Lell Ile Wall Phe SO 55 60
Thir Wall Wall Gly Asn Wall Lell Wall Wall Ile Ala Wall Lell Thir Ser Arg 65 70
Ala Luell Arg Ala Pro Glin Asn Luell Phe Luell Wall Ser Lell Ala Ser Ala 85 90 95
Asp Ile Luell Wall Ala Thir Lell Wall Met Pro Phe Ser Lell Ala Asn Glu 105 11 O
Lell Met Ala Trp Phe Gly Glin Wall Trp Gly Wall Luell 115 12 O 125
Ala Luell Asp Wall Lell Phe Cys Thir Ser Ser Ile Wall His Luell Ala 13 O 135 14 O
Ile Ser Luell Asp Arg Tyr Trp Ser Wall Thir Glin Ala Wall Glu Asn 145 150 155 160
Lell Arg Thir Pro Arg Arg Wall Ala Thir Ile Wall Ala Wall Trp 1.65 17s
Lell Ile Ser Ala Wall Ile Ser Phe Pro Pro Luell Wall Ser Luell Arg 18O 185 19 O
Glin Pro Asp Gly Ala Ala Pro Gly Lell Asn Asp Glu Thir 195
Trp Tyr Ile Luell Ser Ser Cys Ile Ser Phe Phe Ala Pro Luell 21 O 215 22O
Ile Met Gly Luell Wall Tyr Ala Arg Arg Wall Ala Luell Arg 225 23 O 235 24 O
Thir Arg Thir Luell Ser Glu Arg Pro Wall Pro Asp Gly Ala 245 250 255
Ser Pro Thir Thir Glu Asn Gly Luell Ala Ala Gly Ala Gly Glu 26 O 27 O
Asn Gly His Ala Pro Pro Pro Asp Wall Pro Asp Glu Ser 27s 28O 285
Ser Ala Ala Ala Glu Arg Arg Arg Arg Arg Gly Lell Arg Arg Gly 29 O 295
Gly Arg Arg Arg Ala Gly Ala Glu Gly Gly Ala Gly Ala Asp Gly 3. OS 310 315
Glin Gly Ala Gly Pro Gly Ala Ala Glu Ser Gly Lell Thir Ala Ser 3.25 330 335
Arg Ser Pro Gly Pro Gly Gly Arg Luell Ser Arg Ser Ser Arg Ser 34 O 345 35. O
Wall Glu Phe Phe Lell Ser Arg Arg Arg Arg Ala Arg Ser Ser Wall Cys 355 360 365
Arg Arg Wall Ala Glin Ala Arg Glu Arg Phe Thir Phe Wall Luell 37 O 375 38O
Ala Wall Wall Met Gly Wall Phe Wall Luell Trp Phe Pro Phe Phe Phe 385 390 395 4 OO
Ser Ser Luell Tyr Gly Ile Arg Glu Ala Glin Wall Pro Gly 4 OS 41O 415
Pro Luell Phe Lys Phe Phe Phe Trp Ile Gly Asn Ser Ser Luell 425 43 O
Asn Pro Wall Ile Tyr Thir Wall Phe Asn Glin Asp Phe Arg Arg Ser Phe 435 44 O 445
His Ile Luell Phe Arg Arg Arg Arg Arg Gly Phe Arg Glin 450 45.5 460
<210s, SEQ ID NO 7
US 8,080,578 B2 113 114 - Continued agc gca gcg gcc gag agg C9g C9g cgc cgg ggc gcg ttg cgg cgg ggc 912 Ser Ala Ala Ala Glu Arg Arg Arg Arg Arg Gly Ala Lell Arg Arg Gly 29 O 295 3 OO
999 cgg cgg cga gcg gC gC9 gag 999 ggc gC9 ggc ggit gcg gac 999 96.O Gly Arg Arg Arg Ala Gly Ala Glu Gly Gly Ala Gly Gly Ala Asp Gly 3. OS 310 315 32O
Cag 999 gcg gct gag ticg ggg gC9 Ctg acc gcc t cc agg to c cc.g 999 OO8 Glin Gly Ala Ala Glu Ser Gly Ala Luell Thir Ala Ser Arg Ser Pro Gly 3.25 330 335 c cc ggt ggc cgc Ctc. tcg cgc gcc agc tog cgc t cc gtc gag ttic ttic Pro Gly Gly Arg Lieu. Ser Arg Ala Ser Ser Arg Ser Wall Glu Phe Phe 34 O 345 35. O
Ctg tog cgc cgg cgc C9g gC9 C9C agc agc gtg tgc cgc cgc aag gtg 104 Lell Ser Arg Arg Arg Arg Ala Arg Ser Ser Wall Cys Arg Arg Lys Wall 355 360 365 gcc cag gcg cgc gag aag cqc ttic acc titt gtg Ctg gct gtg gt C atg 152 Ala Glin Ala Arg Glu Lys Arg Phe Thir Phe Wall Lell Ala Wall Wall Met 37 O 375 38O ggc gtg ttic gtg ctic togc tigg titc cc c ttic titc tto agc tac agc Ctg 2OO Gly Wall Phe Wall Lieu. Cys Trp Phe Pro Phe Phe Phe Ser Ser Luell 385 390 395 4 OO tac ggc at C tgc cgc gag gcc tic cag gtg coc ggc cc.g citc. ttic aag 248 Gly Ile Cys Arg Glu Ala Cys Glin Wall Pro Gly Pro Luell Phe Lys 4 OS 41O 415 tto ttic ttic tgg atc ggc tac tic aac agc tog citc. aac cc.g gt C at C 296 Phe Phe Phe Trp Ile Gly Tyr Cys Asn Ser Ser Lell Asn Pro Wall Ile 425 43 O tac acg gt C ttic aac cag gat titc cgg cga to c titt aag CaC at C citc. 344 Thir Wall Phe Asn Glin Asp Phe Arg Arg Ser Phe Lys His Ile Luell 435 44 O 445 tto cga cgg agg aga agg ggc titc agg cag tga 377 Phe Arg Arg Arg Arg Arg Gly Phe Arg Glin 450 45.5
<210s, SEQ ID NO 8 &211s LENGTH: 458 212. TYPE : PRT &213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 8
Met Ala Ser Pro Ala Lieu Ala Ala Ala Luell Ala Wall Ala Ala Ala Ala 1. 15
Gly Pro Asn Ala Ser Gly Ala Gly Glu Arg Gly Ser Gly Gly Wall Ala 25 3 O
Asn Ala Ser Gly Ala Ser Trp Gly Pro Pro Arg Gly Glin Ser Ala 35 4 O 45
Gly Ala Wall Ala Gly Lieu Ala Ala Wall Wall Gly Phe Lell Ile Wall Phe SO 55 60
Thir Wall Wall Gly Asn. Wall Lieu Wall Wall Ile Ala Wall Lell Thir Ser Arg 65 70
Ala Luell Arg Ala Pro Glin Asn Lieu. Phe Luell Wall Ser Lell Ala Ser Ala 85 90 95
Asp Ile Luell Wall Ala Thir Lieu Wall Met Pro Phe Ser Lell Ala Asn Glu 105 11 O
Lell Met Ala Trp Tyr Phe Gly Glin Wall Trp Gly Wall Luell 115 12 O 125
Ala Luell Asp Wall Lieu. Phe Cys Thr Ser Ser Ile Wall His Luell Ala 13 O 135 14 O US 8,080,578 B2 115 116 - Continued Ile Ser Lieu. Asp Arg Tyr Trp Ser Wall Thr Glin Ala Val Glu Tyr Asn 145 150 155 160
Lell Arg Thir Pro Arg Arg Wall Lys Ala Thir Ile Wall Ala Wall Trp 1.65 17s
Lell Ile Ser Ala Wall Ile Ser Phe Pro Pro Leu Val Ser Luell Arg 18O 185 19 O
Glin Pro Asp Gly Ala Ala Pro Gly Lell Asn Asp Glu Thir 195
Trp Tyr Ile Luell Ser Ser Cys Ile Ser Phe Phe Ala Pro Luell 21 O 215 22O
Ile Met Gly Lieu Val Tyr Ala Arg Arg Wall Ala Lieu. Arg 225 23 O 235 24 O
Thir Arg Thir Luell Ser Glu Arg Pro Wall Gly Pro Asp Gly Ala 245 250 255
Ser Pro Th Thr Glu Asn Gly Lieu Ala Ala Ala Gly Ala Gly Glu 26 O 27 O
Asn Gly His Ala Pro Pro Pro Asp Wall Glu Pro Asp Glu Ser 27s 28O 285
Ser Ala Ala Ala Glu Arg Arg Arg Arg Arg Gly Ala Lell Arg Arg Gly 29 O 295 3 OO
Gly Arg Arg Arg Ala Gly Ala Glu Gly Gly Ala Gly Gly Ala Asp Gly 3. OS 310 315
Glin Gly Ala Ala Glu Ser Gly Ala Luell Thir Ala Ser Arg Ser Pro Gly 3.25 330 335
Pro Gly Gly Arg Lieu. Ser Arg Ala Ser Ser Arg Ser Wall Glu Phe Phe 34 O 345 35. O
Lieu. Ser Arg Arg Arg Arg Ala Arg Ser Ser Wall Arg Arg Wall 355 360 365
Ala Glin Ala Arg Glu Arg Phe Thir Phe Wall Leu Ala Wall Wall Met 37 O 375
Gly Wall Phe Wall Lieu. Cys Trp Phe Pro Phe Phe Phe Ser Ser Luell 385 390 395 4 OO
Gly Ile Arg Glu Ala Glin Wall Pro Gly Pro Luell Phe 4 OS 415
Phe Phe Phe Trp Ile Gly Asn. Ser Ser Lell Asn Pro Wall Ile 42O 425 43 O
Thir Wall Phe Asn Glin Asp Phe Arg Arg Ser Phe Llys His Ile Lieu. 435 44 O 445
Phe Arg Arg Arg Arg Arg Gly Phe Arg Glin 450 45.5
What is claimed is: 6. The method of claim 1, further comprising ordering a 1. A method for treating a human patient for heart failure, test from a sample from the patient that determines the cardiac arrhythmia, or hypertension with bucindolol compris patient’s genotype. ing: administering bucindolol to the patient if the patient is 55 7. A method for treating a human patient with symptoms of determined to be homozygous wildtype for Del322-325 in the C.2c andrenergic receptor (AR) gene based on a test of a or a diagnosis of heart failure, cardiac arrhythmia, or hyper biological sample from the patient. tension with bucindolol comprising: treating the patient with 2. The method of claim 1, wherein the results are obtained bucindolol if the patient is tested and determined not to be a by receiving a report containing the information or taking a 60 Del322-325 carrier in the C.2c andrenergic receptor (AR) patient history that reveals the results. gene based on results of a test of a biological sample from the 3. The method of claim 1, whereinbucindolol is formulated patient. for oral administration. 8. The method of claim 7, wherein the results are obtained 4. The method of claim 1, wherein the patient is also treated by receiving a report containing the information or taking a with ACE inhibitors, digoxin or a diuretic. 65 patient history that reveals the results. 5. The method of claim 1, wherein bucindolol is adminis 9. The method of claim 7, whereinbucindolol is formulated tered every 12 hours. for oral administration. US 8,080,578 B2 117 118 10. The method of claim 7, wherein the patient is also a) obtaining results from a test that determines whether the treated with ACE inhibitors, digoxin or a diuretic. patient’s genotype is homozygous wildtype for Del322 11. The method of claim 7, wherein bucindolol is admin 325 in the CAR gene; and, b) then treating the patient with bucindolol if the test indi istered or prescribed for administration every 12 hours. cates that the patient’s genotype is homozygous wild 12. The method of claim 7, further comprising ordering a type for Del322-325 in the CAR gene. test from a sample from the patient that determines the 20. The method of claim 19, wherein the results are patient’s genotype. obtained by receiving a report containing the information or 13. A method for administering bucindolol to a human taking a patient history that reveals the results. patient with symptoms of or a diagnosis of heart failure, 10 21. The method of claim 19, wherein bucindolol is formu cardiac arrhythmia, or hypertension comprising: lated for oral administration. a) obtaining information about whether the patient’s geno 22. The method of claim 19, wherein the patient is also type is homozygous wildtype for Del322-325 in the C. treated with ACE inhibitors, digoxin or a diuretic. AR gene; and 23. The method of claim 19, further comprising ordering a b) then administering bucindolol to the patient if the test from a sample from the patient that determines the patient’s genotype is homozygous wildtype for Del322 15 patient’s genotype. 325 in the CAR gene. 24. A method for treating heart failure or cardiac arrhyth 14. The method of claim 13, wherein the results are mia comprising administering bucindolol to a heart failure or obtained by receiving a report containing the information or cardiac arrhythmia human patient who has been determined taking a patient history that reveals the results. to be homozygous wildtype for Del322-325 in the C.2c andr 15. The method of claim 13, wherein bucindolol is formu energic receptor (AR) gene based on a test of a biological lated for oral administration. sample from the patient. 16. The method of claim 13, wherein the patient is also 25. The method of claim 24, wherein bucindolol is formu treated with ACE inhibitors, digoxin or a diuretic. lated for oral administration. 17. The method of claim 13, wherein bucindolol is admin 25 26. The method of claim 24, wherein the patient is also istered every 12 hours. treated with ACE inhibitors, digoxin or a diuretic. 18. The method of claim 13, wherein the information is 27. The method of claim 24, wherein bucindolol is admin obtained by ordering a test from a sample from the patient that istered every 12 hours. determines the patient’s genotype. 28. The method of claim 24, further comprising ordering a 19. A method for treating a human patient for heart failure, 30 test using a sample from the patient that determines the cardiac arrhythmia, or hypertension with bucindolol compris patient’s genotype. 1ng: UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,080,578 B2 Page 1 of 1 APPLICATIONNO. : 1 1/8381.31 DATED : December 20, 2011 INVENTOR(S) : Liggett et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
On the Title Page:
The first or Sole Notice should read --
Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 300 days.
Signed and Sealed this Seventeenth Day of April, 2012
David J. Kappos Director of the United States Patent and Trademark Office