Methods and compositions involving bucindolol for the treatment of heart disease

Abstract

The current methods and compositions relate, in certain aspects, to treatment of atrial fibrillation with bucindolol in patients, including patients with heart failure, after being determined to have non-internalizing ß-adrenergic receptors.

Description

DESCRIPTIONMETHODS AND COMPOSITIONS INVOLVING BUCINDOLOL FOR THE TREATMENT OF HEART DISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority and the benefit of U.S. Provisional Application No. 63 / 650,312 filed May 21, 2024 which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION1. Field of the Invention

[0002] The present disclosure relates generally to the fields of molecular biology, cardiology, and medicine.2. Background

[0003] Atrial fibrillation (AF) is a serious cardiovascular condition associated with increased risks for stroke,3development4or worsening4-6of heart failure (HF), ventricular arrhythmias and sudden cardiac death,7decreased exercise tolerance,8and a variety of potentially disabling symptoms.9,10The most recent estimate of the AF prevalence in the U.S. is in the 3-6 million range,11projected to increase to 6-16 million cases by 2050.12Of comorbidities that commonly occur with AF, HF is associated with a prevalence varying from 21% to 68%12and is the largest contributor to mortality risk.13Drug therapy to prevent recurrent AF in HF patients consists of ion channel acting agents that are only modestly effective, and in HF patients are associated with significant adverse event profiles including precipitation of serious ventricular arrhythmias.14,15A more effective treatment for AF is catheter ablation, but cost, technical challenges and a relatively high eventual failure rate confines this treatment to only a minority of patients.16AF pathophysiology consists of structural and molecular atrial remodeling resulting in dilated, fibrotic and hypocontractile atrial chambers that exhibit increased electrical automaticity, shortened refractoriness, and other electrophysiologic abnormalities.17This pathophysiology is similar to LV pathologic remodeling in HF, unsurprising because of their overlapping risk factors. No drug approved for AF has a favorable effect on HF, and most are capable of exacerbating myocardialdysfunction and clinical HF symptoms. Taken together, these observations define a serious unmet therapeutic need for AF prevention in HF (AF / HF) patients.18

[0004] This problem is best addressed by the successful development of a drug that prevents AF via a favorable biologic effect on both the pathologically remodeled LA and LV.SUMMARY OF THE DISCLOSURE

[0005] The inventors provide treatment methods that meet the needs described above. Described herein are ways to provide therapeutic benefits to patients with heart failure, atrial fibrillation, other cardiac arrhythmia, and other cardiovascular conditions and diseases through the identification of those patients whose therapeutic outcome can be achieved with bucindolol, including particular dosage regimens of bucindolol. The identification of the patients can be achieved by genotyping the patients and identifying specific genotypes and / or haplotypes that correspond to better treatment outcomes. Aspects herein include, among others, investigations designed to further the scientific basis for how such a drug successfully prevents atrial fibrillation and treats heart failure in a genetically defined heart failure population, while seeking to refine and expand the pharmacogenetic scope of the target population.

[0006] Disclosed are methods of treating, preventing, delaying onset of, and / or reducing the risk of atrial fibrillation in a patient. In certain aspects, the methods favorably affect the natural history of heart failure in a patient. In certain aspects, the atrial fibrillation is new onset atrial fibrillation. In certain aspects, the atrial fibrillation is recurrent atrial fibrillation. The method can comprise one or more steps including administering an effective amount of bucindolol to a patient after the patient has been genotyped for one or more B-adrenergic receptors. The method can also comprise one or more steps including genotyping the patient, sequencing nucleic acid from a biological sample from the patient, analyzing sequencing data, and / or monitoring the patient. In certain aspects, the method comprises or consists of administering an effective amount of bucindolol to a patient determined to have a particular genotype, as discussed herein.

[0007] In certain aspects, the patient has been genotyped and found to have at least one copy of a non-internalizing B-adrenergic receptor. A non-internalizing B-adrenergic receptor can be a B-adrenergic receptor comprising one or more polymorphisms that reduces or prevents internalization by normal cellular machinery, such as by B-arrestin 1 and / or B-arrestin 2. The non-internalizing B-adrenergic receptor can include B-adrenergic receptors that do notinternalize after phosphorylation, including phosphorylation by G-protein receptor kinases. In certain aspects, at least one of the non-internalizing B-adrenergic receptors comprises a Bl- adrenergic receptor (BiAR) having a serine at amino acid position 49. In certain aspects, at least one of the non-internalizing B-adrenergic receptors comprises a B2-adrenergic receptor (B2AR) that does not have a glutamine at position 27 and an arginine at position 16 (z.e., lacks a Gln27Argl6 haplotype). In certain aspects, the patient was found to have at least two copies of a non-internalizing B-adrenergic receptor. In certain aspects, the patient was found to have at least two copies of a Bl-adrenergic receptor having a serine at amino acid position 49. In certain aspects, the patient was further found to have a B2-adrenergic receptor haplotype other than Gln27Argl6. In certain aspects, the patient was further found to have at least one copy of a Bl- adrenergic receptor with an arginine at amino acid position 389. In certain aspects, the patient was further determined to have at least two copies of a Bl-adrenergic receptor with an arginine at amino acid position 389.

[0008] In certain aspects, the patient is administered an effective amount of bucindolol after being genotyped. The effective amount may be determined by one skilled in the art. In certain aspects, the patient is administered about 12.5 to 200 mg of bucindolol per day. In certain aspects, the patient is administered 12.5 to 200 mg of bucindolol per day. In certain aspects, the patient is administered at least, at most, or about 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23,23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33,33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43,43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53,53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63,63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 70.5, 71, 71.5, 72, 72.5, 73,73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80, 80.5, 81, 81.5, 82, 82.5, 83,83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93,93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 100, 100.5, 101, 101.5, 102,102.5, 103, 103.5, 104, 104.5, 105, 105.5, 106, 106.5, 107, 107.5, 108, 108.5, 109, 109.5, 110,110.5, 111, 111.5, 112, 112.5, 113, 113.5, 114, 114.5, 115, 115.5, 116, 116.5, 117, 117.5, 118,118.5, 119, 119.5, 120, 120.5, 121, 121.5, 122, 122.5, 123, 123.5, 124, 124.5, 125, 125.5, 126,126.5, 127, 127.5, 128, 128.5, 129, 129.5, 130, 130.5, 131, 131.5, 132, 132.5, 133, 133.5, 134,134.5, 135, 135.5, 136, 136.5, 137, 137.5, 138, 138.5, 139, 139.5, 140, 140.5, 141, 141.5, 142,142.5, 143, 143.5, 144, 144.5, 145, 145.5, 146, 146.5, 147, 147.5, 148, 148.5, 149, 149.5, 150,150.5, 151, 151.5, 152, 152.5, 153, 153.5, 154, 154.5, 155, 155.5, 156, 156.5, 157, 157.5, 158,158.5, 159, 159.5, 160, 160.5, 161, 161.5, 162, 162.5, 163, 163.5, 164, 164.5, 165, 165.5, 166,166.5, 167, 167.5, 168, 168.5, 169, 169.5, 170, 170.5, 171, 171.5, 172, 172.5, 173, 173.5, 174,174.5, 175, 175.5, 176, 176.5, 177, 177.5, 178, 178.5, 179, 179.5, 180, 180.5, 181, 181.5, 182,182.5, 183, 183.5, 184, 184.5, 185, 185.5, 186, 186.5, 187, 187.5, 188, 188.5, 189, 189.5, 190,190.5, 191, 191.5, 192, 192.5, 193, 193.5, 194, 194.5, 195, 195.5, 196, 196.5, 197, 197.5, 198,198.5, 199, 199.5, or 200 mg of bucindolol, or any range derivable therein, per day. In certain aspects, the patient is administered about 0.15 to 2.5 mg / kg of bucindolol per day. In certain aspects, the patient is administered at least, at most, or about 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73,1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9,1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.1, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.2, 2.21, 2.22, 2.23, 2.24,2.25, 2.26, 2.27, 2.28, 2.29, 2.3, 2.31, 2.32, 2.33, 2.34, 2.35, 2.36, 2.37, 2.38, 2.39, 2.4, 2.41,2.42, 2.43, 2.44, 2.45, 2.46, 2.47, 2.48, 2.49, or 2.5 mg / kg of bucindolol, or any range derivable therein, per day.

[0009] Also disclosed are methods of treating, preventing, delaying onset of, or reducing the risk of cardiovascular mortality, methods of treating, preventing, delaying onset of, or reducing the risk of heart failure hospitalization, methods of treating, preventing, delaying onset of, or reducing the risk of all-cause mortality in a patient, such as a heart failure patient. In certain aspects, the patient has, is suspected of having, or has been diagnosed with having heart failure. In certain aspects, the heart failure is heart failure with preserved ejection fraction (HFpEF). In certain aspects, the heart failure is heart failure with preserved ejection fraction (HFpEF). In certain aspects, the heart failure is heart failure with mid-range ejection fraction (HFmrEF). In certain aspects, the heart failure is heart failure with reduced ejection fraction(HFrEF). In certain aspects, the patient has a left ventricular ejection fraction (LVEF) of less than or equal to 35%, or any range derivable therein. In certain aspects, the patient has a left ventricular ejection fraction (LVEF) of greater than, at most, or equal to 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, or any range derivable therein. Also disclosed are methods of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, cardiovascular mortality, heart failure hospitalization, and / or all-cause mortality in a patient, the method comprising administering an effective amount of bucindolol to a patient identified as having or determined to have an ADRB1 Arg389Ser49 haplotype. Also disclosed are methods of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, cardiovascular mortality, heart failure hospitalization, and / or all-cause mortality, the method comprising administering an effective amount of bucindolol to a heart failure patient identified as having an ADRB1 Gly389Ser49 haplotype. In certain aspects, the patient is further identified as having two copies of an ADRB1 Ser49 gene. In certain aspects, the patient is further identified as having no detectable copies of a Gln27Argl6 haplotype. Genotyping, used in certain aspects, is physical and / or chemical alteration of a nucleic acid to determine its sequence at a particular location(s). In certain aspects, genotyping comprises sequencing, nucleic acid amplification, hybridization, and / or transcription, or a combination thereof.

[0010] Also disclosed are methods of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering 12.5 to 200 mg of bucindolol to a patient who has been genotyped and found to have at least one copy of an ADRB1 Gly389Ser49 haplotype and no copies of an ADBR2 Gln27Argl6 haplotype.

[0011] Also disclosed are methods of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering 12.5 to 200 mg of bucindolol to a patient who has been genotyped and found to have at least one copy of an ADRB1 Arg389Ser49 haplotype and no copies an ADBR2 Gln27Argl6 haplotype.

[0012] Also disclosed are methods of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering 12.5 to 200 mg of bucindolol to a patient who has been genotyped and found to have no copies of an ADBR2 Gln27Argl6 haplotype.

[0013] In certain aspects, a composition is a racemic mixture of S-bucindolol and R- bucindolol. In other aspects, it comprises substantially more of one enantiomer than the other. In specific aspects, a composition comprises a ratio of S-bucindolol to R-bucindolol of at least 99: 1 by weight of bucindolol in the composition.

[0014] Presentation of heart failure may include or exclude at least 1, 2, 3, 4, 5, or 6 of the following signs or symptoms: breathlessness, exertional dyspnea, orthopnea, paroxysmal nocturnal dyspnea, dyspnea at rest, or acute pulmonary edema. There may be other cardiac symptoms of heart failure such as chest pain / pressure and palpitations. Common noncardiac signs and symptoms of heart failure include anorexia, nausea, weight loss, bloating, fatigue, weakness, oliguria, nocturia, and cerebral symptoms of varying severity, ranging from anxiety to memory impairment and confusion. Findings from the Framingham Heart Study supported the idea that subclinical cardiac dysfunction and noncardiac comorbidities are associated with increased incidence of heart failure, supporting the concept that heart failure is a progressive syndrome and that noncardiac factors are very relevant. Presentation of cardiac arrhythmia may be, be at most, or be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the following signs or symptoms: palpitations, dyspnea, fatigue, dizziness, angina, decompensated heart failure, hemodynamic dysfunction, tachycardia-induced cardiomyopathy, and systemic thromboembolism. In some aspects, methods include or exclude alleviating, addressing or reducing symptom burden in a heart failure and / or atrial fibrillation patient. Symptoms that may be included as symptom burden for atrial fibrillation ban exclude or include palpitations, chest pain, dizziness, fainting, irregular pulse, palpitations, shortness of breath, sweating, and / or tiredness.

[0015] To achieve these methods, a doctor, medical practitioner, or their staff may obtain a biological sample from the patient for evaluation. The sample may be analyzed by the practitioner or their staff, or it may be sent to an outside or independent laboratory. The medical practitioner may be cognizant of whether the test is providing information regarding the patient’s piAR and / or P2AR genes, or the medical practitioner may be aware only that the test indicates directly or indirectly that the genotype of the patient reflects a particular genotype or genotypes.

[0016] In any of these circumstances, the medical practitioner “knows” or identifies indirectly the relevant information that will allow him or her to determine whether bucindolol is an appropriate medical treatment. It is contemplated that, for example, a laboratory conducts the test to determine that patient’s genotype such its personnel also know the appropriateinformation. They may report back to the practitioner with the specific result of the test performed or the laboratory may simply report that bucindolol is an appropriate drug based on the laboratory results. Moreover, through these different channels, the patient’s genotype at any particular polymorphism and / or genotype can be known.

[0017] Certain aspects are directed to a tangible, computer-readable medium comprising a genotype profile of a subject, wherein the genotype or haplotype profile exhibits the sequence at one or more polymorphisms in one or both alleles of the piAR gene and / or a P2AR gene. In certain aspects the medium comprising the genotype profile of the subject exhibits the patient is homozygous Arg389 in the PiAR gene. In certain aspects the medium comprising the genotype profile of the subject exhibits the patient is heterozygous Arg389 in the piAR gene. In certain aspects the medium comprising the genotype profile of the subject exhibits the patient is homozygous Ser49 in the PiAR gene. In certain aspects the medium comprising the genotype profile of the subject exhibits the patient is heterozygous Ser49 in the piAR gene. In certain aspects the medium comprising the genotype profile of the subject exhibits the patient is homozygous Glu27 in the P2AR gene. In certain aspects the medium comprising the genotype profile of the subject exhibits the patient is heterozygous Glu27 in the P2AR gene.

[0018] In certain aspects the medium comprising the haplotype profile of the subject exhibits two copies of the ADRB1 Arg389Ser49 haplotype, which can be detected by determining the individual is homozygous for ADRB1 Arg389 and Ser49 genotypes. In certain aspects the medium comprising the haplotype profile of the subject exhibits two copies of the ADRB1 Arg389Ser49 haplotype and one copy of the ADRB2 Glu27 genotype. In certain aspects of the medium comprising the haplotype profile of the subject exhibits two copies of the ADRB1 Gly389Ser49 haplotype, which can be detected by determining the individual is homozygous for ADRB1 Gly389 and Ser49 genotypes. In certain aspects of the medium comprising the haplotype profile of the subject exhibits two copies of the ADRB1 Gly389Ser49 haplotype, plus one copy of the ADRB2 Glu27 genotype.

[0019] In some aspects, genotyping and / or haplotyping is done by DNA amplicon sequencing. In certain aspects, the DNA amplicon sequencing comprises amplifying a region corresponding to amino acid 389 of ADBR1, which may be nucleotide position 1165 on the ADBR1 gene. In certain aspects, the DNA amplicon sequencing comprises amplifying a region corresponding to amino acid 49 of ADBR1, nucleotide position 145 on the ADBR1 gene. In some aspects, the haplotyping is done by long-read sequencing.

[0020] In some aspects, the patient was initially diagnosed with heart failure less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the administering, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months before the administering, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 90, 120, 150, or 180 days before the administering. In some aspects, the patient initially developed heart failure less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the administering, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months before the administering, or less than 30, 60, 90, 120, 150, or 180 days before the administering.

[0021] As used herein, a patient is “initially diagnosed” with a disease when the patient is diagnosed with the disease for the first time. As used herein, a diagnosis occurs at the time that a healthcare provider, after concluding that the patient has a disease or condition, either (1) documents such conclusion in a medical record, (2) communicates to the patient such conclusion, or (3) prescribes a medication to treat the disease or condition.

[0022] In some aspects, the patient has further been diagnosed with atrial fibrillation or atrial flutter. In certain aspects, the patient has not had atrial fibrillation for more than two years before receiving bucindolol. In certain aspects, the patient has not had atrial fibrillation for more than two years before bucindolol is administered to the patient. In certain aspects, atrial fibrillation is not present for more than two years before being diagnosed with heart failure. In some aspects, the patient has not had atrial fibrillation or heart failure for more than 12 years. In some aspects, the patient was initially diagnosed with atrial fibrillation or atrial flutter approximately or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years before the administering (or any range derivable therein), or between or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months (or any range derivable therein) before the administering, or less than 30, 60, 90, 120, 150, or 180 days before the administering of bucindolol. In some aspects, the patient developed or was initially diagnosed atrial fibrillation or atrial flutter less than 12 years before administering bucindolol. In some aspects, a patient has been diagnosed with both heart failure and atrial fibrillation (or atrial flutter). In some aspects, the patient initially developed atrial fibrillation or atrial flutter less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the administering, or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months before the administering, or less than 30, 60, 90, 120, 150, or 180 days before the administering. In some aspects in which thepatient has been diagnosed with both heart failure and atrial fibrillation, the respective diagnoses occurred contemporaneously or within a relatively short time of each other. In some aspects, the patient was diagnosed with heart failure prior to developing atrial fibrillation. In some aspects, the patient was initially diagnosed with atrial fibrillation before or < 2 years after initially being diagnosed with heart failure. In some aspects, the patient was initially diagnosed with atrial fibrillation before or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months (or any range derivable therein) before being diagnosed with heart failure.

[0023] In certain aspects, the total symptom burden of AF is measured in the patient. In certain aspects, an effective amount of bucindolol is administered to a patient with a specific total symptom burden.

[0024] In some aspects, the patient has a history of atrial fibrillation but is in sinus rhythm at the time that bucindolol is administered.

[0025] In some aspects, the patient is administered about 12.5 to 200 mg of bucindolol per day. In some aspects, the patient is administered at least, at most, or 5, 7.5, 10, 12.5, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 mg of bucindolol per day, or between any two of these values. In some aspects, the patient is administered bucindolol in a dosage of 0.15 to 2.5 mg / kg per day. In some aspects, the patient is administered bucindolol in a dosage of at least, at most, or 0.05, 0.10, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 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, 2.0,2.1, 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, 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, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 mg / kg per day, or between any two of these values.

[0026] In any method disclosed herein in which a patient’s characteristics are described, such as a patient having a disease or condition (e.g., heart failure, atrial fibrillation, an LVEF value, a genotype, etc.), it is contemplated that an aspect of such a method may include administering an effective amount of bucindolol to a patient that has the disease or condition or to a patient that has been diagnosed with the disease or condition. It is further contemplated that aspects of such methods may additionally or alternatively include administering bucindolol to a patient after determining or ascertaining that the patient has the disease or condition or has been diagnosed with the disease or condition. It is still further contemplated that aspects mayalso additionally or alternatively include first obtaining the results of a test or evaluation that shows or indicates that the patient has the disease or condition, and administering bucindolol to the patient only after obtaining such results.

[0027] Multiple aspects of the invention are discussed throughout this application. Any aspect discussed with respect to one aspect applies to other aspects as well and vice versa. Each aspect described herein is understood to be applicable to all aspects. It is contemplated that any aspect discussed herein can be implemented with respect to any method or composition, and vice versa. For example, in describing certain aspects herein, there are values disclosed such as time from diagnosis of a disease or condition, time between diagnoses of heart failure and atrial fibrillation, LVEF values, timing of EVEF testing and testing for arrhythmia, and dosage of bucindolol, among others. Various patient characteristics, such as having or having been diagnosed with a certain disease or condition, are also disclosed in certain aspects. It is contemplated that the values, patient characteristics, and other features discussed in the context of one aspect can also be incorporated into any other aspect disclosed herein. Furthermore, compositions and kits can be used to achieve methods disclosed herein.

[0028] In aspects of the methods involving administering bucindolol disclosed herein, the administering has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (or any range derivable therein) of the following effects: reduced risk of all-cause mortality; reduced risk death caused by heart failure; reduced risk of hospitalization; reduced risk of hospitalization for heart failure; reduced hospitalization for heart failure; reduced risk of stroke; reduced risk of new onset or recurrent atrial fibrillation, atrial flutter, ventricular tachycardia, ventricular fibrillation, tachycardia, or cardiac arrhythmia; reduced risk of needing therapies such as cardiac ablation or anti- arrhythmic medication; increased delay in onset of new onset or recurrent atrial fibrillation or atrial flutter; reduction or elimination of one or more symptoms, alleviation or reduction of symptom burden or improved quality of life. In some aspects in which a reduced risk of an event or condition is achieved, the risk is reduced by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%. In some aspects in which the length of a hospital stay is reduced, the hospital stay is reduced by at least or by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days (or any range derivable therein).

[0029] The terms “effective amount” or “therapeutically effective amount” refer to that amount of a composition of the disclosure that is sufficient to effect treatment, as definedherein, when administered to a mammal in need of such treatment. This amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular composition of the disclosure chosen, the dosing regimen to be followed, timing of administration, manner of administration and the like, all of which can readily be determined by one of ordinary skill in the art.

[0030] The “numerical values” and “ranges” provided for the various substituents are intended to encompass all integers within the recited range.

[0031] The term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.

[0032] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. In several aspects, these media and agents can be used in combination with pharmaceutically active substances. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0033] The term “treatment” or “treating” means any treatment of a disease or disorder in a mammal, including inhibiting the disease or disorder, arresting or suppressing the development of clinical symptoms; improving one or more physiological effects of the disease or disorder; reducing the severity or risk of an adverse event; reducing mortality from the disease or disorder; reducing the risk of mortality from the disease or disorder; reduced need for additional or alternative therapies such as cardiac ablation or use of anti- arrhythmic medication due to the disease or disrder; reduced risk of new-onset or recurrent atrial fibrillation, atrial flutter, ventricular tachycardia, ventricular fibrillation, or cardiac arrhythmia associated with the disease or disorder; reduced risk of hospitalization from the disease or disorder; reduced time of hospitalization from the disease or disorder; an increase in the delay of the onset of one or more symptoms or disorders associated with or caused by the disease or disorder; relieving the disease or disorder; and / or causing the regression of clinical symptoms from the disease or disorder.

[0034] Methods may involve multiple administrations of one or more compounds, compositions, and / or agents. In certain aspects, cells or a subject are provided with a tolerance inducing agent prior to administering the composition for which a tolerance is being induced. It is contemplated that compounds, compositions, and / or agents may be formulated in a pharmaceutically acceptable formulation in certain aspects of the invention.

[0035] Also disclosed are any of the following enumerated aspects:1. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering an effective amount of bucindolol to a patient who has been genotyped and found to have at least one copy of a non-internalizing B-adrenergic receptor.2. The method of aspect 1, wherein the atrial fibrillation is new onset atrial fibrillation.3. The method of aspect 1 or 2, wherein the atrial fibrillation is recurrent atrial fibrillation.4. The method of any one of aspects 1 to 3, wherein the patient has, is suspected of having, or has been diagnosed with having heart failure.5. The method of any one of aspects 1 to 4, wherein at least one of the noninternalizing B-adrenergic receptors comprises a Bl -adrenergic receptor having a serine at amino acid position 49.6. The method of any one of aspects 1 to 5, wherein the patient has been found to have two copies of a Bl -adrenergic receptor having a serine at amino acid position 49.7. The method of any one of aspects 1 to 5, wherein at least one of the noninternalizing B-adrenergic receptors comprises a B2-adrenergic receptor not having a Gln27Argl6 haplotype.8. The method of any one of aspects 1 to 7, wherein the patient has been found to have one copy of a B2-adrenergic receptor that is not a Gln27Argl6 haplotype.9. The method of any one of aspects 1 to 7, wherein the patient has been found to have two copies of a B2-adrenergic receptor that is not a Gln27Argl6 haplotype.10. The method of any one of aspects 1 to 9, wherein the patient was further found to have at least one copy of a Bl -adrenergic receptor having an arginine at amino acid position389.11. The method of any one of aspects 1 to 10, wherein the patient was further found to have at least one copy of a Bl -adrenergic receptor having a glycine at amino acid position 389.12. The method of any one of aspects 1 to 11, wherein the patient was further found to have at least two copies of a Bl-adrenergic receptor having an arginine at amino acid position 389.13. The method of any one of aspects 1 to 12, wherein the patient is administered about 12.5 to 200 mg of bucindolol per day.14. The method of any one of aspects 1 to 13, wherein the patient is administered about 0.15 to 5 mg / kg of bucindolol per day.15. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of cardiovascular mortality, the method comprising administering an effective amount of bucindolol to a patient who has been genotyped and found to have at least one copy of a non-internalizing B-adrenergic receptor.16. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of heart failure hospitalization, the method comprising administering an effective amount of bucindolol to a patient who has been genotyped and found to have at least one copy of a non-internalizing B-adrenergic receptor.17. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of all-cause mortality, the method comprising administering an effective amount of bucindolol to a patient who has been genotyped and found to have at least one copy of a non-internalizing B-adrenergic receptor.18. The method of any one of aspects 15 to 17, wherein the patient has, is suspected of having, or has been diagnosed with having heart failure.19. The method of any one of aspects 15 to 18, wherein at least one of the noninternalizing B-adrenergic receptors comprises a Bl -adrenergic receptor having a serine at amino acid position 49.20. The method of any one of aspects 15 to 19, wherein the patient has been found to have two copies of a Bl-adrenergic receptor having a serine at amino acid position 49.21. The method of any one of aspects 15 to 20, wherein at least one of the noninternalizing B-adrenergic receptors is not a B2-adrenergic receptor having a Gln27Argl6 haplotype.22. The method of any one of aspects 15 to 21, wherein the patient was found to have at least two copies of a non-internalizing B-adrenergic receptor.23. The method of any one of aspects 15 to 22, wherein the patient was found to have at least two copies of a Bl-adrenergic receptor having a serine at amino acid position 49.24. The method of any one of aspects 15 to 23, wherein the patient was further found to have no copies of a B2-adrenergic receptor having a Gln27Argl6 haplotype.25. The method of any one of aspects 15 to 24, wherein the patient was further found to have at least one copy of a Bl-adrenergic receptor having an arginine at amino acid position 389.26. The method of any one of aspects 15 to 25, wherein the patient was further found to have at least two copies of a Bl-adrenergic receptor having an arginine at amino acid position 389.27. The method of any one of aspects 15 to 26, wherein the patient is administered about 12.5 to 200 mg of bucindolol per day.28. The method of any one of aspects 15 to 27, wherein the patient is administered about 0.15 to 5 mg / kg of bucindolol per day.29. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering an effective amount of bucindolol to a patient identified as having at least one copy of an ADRB1 Arg389Ser49 haplotype.30. The method of aspect 29, wherein the patient is identified as having two copies of an ADRB1 Arg389Ser49 haplotype.31. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering an effective amount of bucindolol to a patient identified as having at least one copy of an ADRB1 Gly389Ser49 haplotype.32. The method of aspect 31, wherein the patient is identified as having two copies of an ADRB1 Gly389Ser49 haplotype.33. The method of any one of aspects 29 to 32, wherein the patient is further identified as having two copies of an ADRB 1 Ser49 genotype.34. The method of any one of aspects 29 to 33, wherein the patient is further identified as having no copies of an ADBR2 Gln27Argl6 haplotype.35. The method of any one of aspects 29 to 34, wherein the patient has, is suspected of having, or has been diagnosed with having heart failure.36. The method of any one of aspects 29 to 35, wherein the patient is administered about 12.5 to 200 mg of bucindolol per day.37. The method of any one of aspects 29 to 36, wherein the patient is administered about 0.15 to 5 mg / kg of bucindolol per day.38. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering 12.5 to 200 mg of bucindolol to a patient who has been genotyped and found to have at least one copy of an ADRB 1 Gly389Ser49 haplotype and no copies of an ADBR2 Gln27Argl6 haplotype.39. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering 12.5 to 200 mg of bucindolol to a patient who has been genotyped and found to have at least one copy of an ADRB 1 Arg389Ser49 haplotype and no copies of an ADBR2 Gln27Argl6 haplotype.40. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering 12.5 to 200 mg ofbucindolol to a patient who has been genotyped and found to have no copies of an ADBR2 Gln27Argl6 haplotype.41. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering an effective amount of a biased-ligand to a patient whose genotype of a B-adrenergic receptor is known.42. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering an effective amount of a beta- AR antagonist to a patient whose genotype of a B-adrenergic receptor is known.43. The method of aspect 42, wherein the genotype is an internalizing B-adrenergic receptor.44. An aspect directed to a method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering an effective amount of a neutral beta- AR agonist to a patient whose genotype of a B-adrenergic receptor is known.

[0036] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0037] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0038] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” “(x and z) or y,” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspect.

[0039] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as“contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0040] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0041] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0042] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0043] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0044] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Brief Description of the Drawings, Detailed Description, and / or Claims.

[0045] The references to the methods of treatment by therapy or surgery or in vivo diagnosis methods in example 1 or any other example of this description and in the claims and disclosure of this description are to be interpreted as references to compounds, pharmaceutical compositions and medicaments of the present invention for use in those methods.

[0046] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0048] FIG. 1 shows a Forest plot of ADRB1, ADRB2 haplotypes (Column 1) event rates (Column 6) and between haplotype groups event rate Odds Ratios (ORs, column 7) in All LVEF cohort patients randomized to placebo in the BEST Adrenergic Receptor Polymorphism substudy. The ADRB1, ADRB2 haplotypes are displayed by ">3 Double Internalizing" combinations in Tier 1 (>3DI, 1 or 2 copies of ADRB1 Arg or Gly389Gly49 AND ADRB2 Gln27Argl6, for a total of 3-4 copies of an internalizing haplotype); "Incompletely Internalizing" in Tier 2 (21), 2 total copies of internalizing haplotypes, either 1 copy of ADRB1 AND ADRB2, or 2 copies on one of them); and ">3 Double Non-internalizing" haplotype combinations in Tier 3 (>3DNI, 0-1 total copies of ADRB1 Arg or Gly389Gly49 AND ADRB2 Gln27Argl6, 3-4 copies of non-internalizing haplotypes). The copy number in Column 1 designates the number of the specific haplotypes in the diplotype: Haplotype combinations in Column 1 follow "AND" logic, meaning both the ADRB1 and ADRB2 haplotypes must be present in the form designated. The counterpart, comparison haplotypes used to generate ORs follow OR logic, i.e. if either the ADRB1 or the ADRB2 haplotype doesn't match the cognate Column 1 haplotype then the pair becomes a member of the counterpart group. Column 5 lists the event rate and [N] in the paired haplotypes in column 1, followed by the event rate and [N] in the counterpart, comparator groups. CVM=Cardiovascular Mortality; AF / AFL / ACM=Atrialfibrillation, Atrial Flutter or All-Cause Mortality; CVM / HFH=CVM or Heart Failure hospitalization; HFH=Heart Failure Hospitalization; ACM / Tx=ACM or Cardiac Transplantation; CVH=Cardiovascular Hospitalization; ACM=A11-Cause Mortality. For the AF / AFL / ACM endpoint, only non-AF patients at the time of randomization are analyzed, resulting in a smaller N than for the 6 HF endpoints. For all endpoints only first events are considered. Odds Ratios (ORs) are calculated by: [(Index event rate in %) / (100 - Index event rate)] / [(Comparator event rate) / (100 - Comparator event rate)], or in Row 1 (8.3 / 91.7) / (18.8 / 81.2) = 0.39.

[0049] FIG. 2 shows the BEST substudy patients randomized to bucindolol in the BEST Adrenergic Receptor Polymorphism substudy. The forest plot is constructed as for FIG. 1.

[0050] FIG. 3 shows a Forest plot of bucindolol vs. placebo treatment effect Odds Ratios (ORs) by haplotype. [Ns] are the number of subjects in the bucindolol and placebo groups respectively; otherwise set-up is as in Figures 1 and 2.

[0051] FIG. 4 shows the time to first event curves in bucindolol vs. placebo treated patients with Maximum Double Non-internalizing (4DNI), total of 4 non-internalizing haplotypes), LVEF >0.20 cohort N=184. A. All-cause Mortality or Cardiac Transplantation (ACM / Tx); B. Heart Failure Hospitalization (HFH); C. Cardiovascular Mortality or Heart Failure Hospitalization (CVM / HFH); D. Cardiovascular Hospitalization (CVH).

[0052] FIG. 5 shows the time to first event curves curves in Blacks or non-Blacks in the All LVEF cohort for the CVM / HFH (A) and CVH (B) time to first event endpoints, with haplotype categories of: Double Internalizing (DI) = >1 ADRB1 AND ADRB2 internalizing haplotypes (ADRB1 Arg389Gly49, ADRB2 Gln27Argl6); Double non-internalizing (NDI) = all other haplotypes.

[0053] FIG. 6 shows the GENETIC-AF study, A. NT-proBNP change from baseline for subjects with >2 double internalizing ADRB1 AND ADRB2 haplotypes (>2DNI) or >2 Noninternalizing haplotypes (>2NI) in the Bucindolol or Metoprolol Groups; B. Same scheme for change from baseline in norepinephrine. Baseline values, changes by diplotype and 4DNI data are in FIG. 17-20.

[0054] FIG. 7 shows subject characteristics by diplotype, (Mean+SD), BEST Adrenergic Receptor Polymorphisms Substudy (N= 1029 ADRB1, 1040 ADRB2). *P<0.05, **P<0.01,***P<0.001 vs. all other members of the same genotype group. In sequence: White / Black / Hispanic / Asian / Pacific Islander, American Indian, Alaskan / Other; *VOL OL (Volume overloaded). M / F refers to male or female self-identified gender. HF=heart failure, HxDM = history of diabetes, Hx Htn=history of hypertension, HR=heart rate, SBP=systolic blood pressure, PNE=plasma norepinephrine, AF= atrial fibrillation.

[0055] FIG. 8 shows minor allele frequencies (MAF) of ADRB1 and ADRB2 polymorphisms by Black, non-Black race; BEST Adrenergic Receptor Polymorphisms Substudy (from genotype data). ^Chi-square 2-sided test (Fisher exact test substituted for low cell counts) performed on the number of alleles.

[0056] FIG. 9 shows Diplotypes, Genotypes BEST Adrenergic Receptor Polymorphisms Substudy. HMZG = homozygote; 1HTZG = single heterozygote; 2HTZG = double heterozygote.

[0057] FIG. 10 shows Linkage Disequilibrium (LD) Coefficients for ADRB1, ADRB2 polymorphisms in the BEST Substudy.

[0058] FIG. 11 shows Haplotype frequencies by Race, BEST Substudy, f = haplotype frequency (haplotype N / Total N Haplotypes).

[0059] FIG. 12 shows summary statistics, from Figures 1-3 and 23-25. Tiers of bi (ADRB1)- and bi (ADRB 2)-adrenergic receptor genes contain overlapping subjects. *Event Rates are mean+SD, Odds Ratios (OR)s are mean (95% CI); For AF / AFL / ACM All LVEF Ns are 403 and 390 for placebo and bucindolol, respectively; *Wilcoxon signed rank Pbo vs. Buc Event Rate.

[0060] FIG. 13 shows By-subject analysis of {ADRB1 / ADRB2} haplotype internalization tier data in Figures 1-3, 23-25, with event probability in tiers ordered from most (Tier 1, >3DI, 3-4 internalizing haplotypes) to least internalizing (Tier 3,>3DNI, 0-1 internalizing haplotypes). *From Tier 1 to Tier 3; 'Chi Square for endpoint slope differences; *Chi Square for unequal slopes between placebo and bucindolol.

[0061] FIG. 14 shows Subject characteristics by diplotype, (Mean+SD), GENETIC-AF Substudy (N = 138). *P<0.05, **P<0.01, ***P<0.001 vs. all other members of the same genotype group. In sequence: White / Black / Hispanic / Asian / Pacific Islander, American Indian,Alaskan / Other; *Paroxysmal / Persistent. HF=heart failure, HxDM = history of diabetes, Hx Htn=history of hypertension, HR=heart rate, SBP=systolic blood pressure, AF= atrial fibrillation.

[0062] FIG. 15 shows Genotypes, haplotypes, and diplotypes in the GENETIC-AF Trial.

[0063] Fig. 16 shows NT -proBNP (pg / ml), Bucindolol Arm. *WSR=Wilcoxon signed rank test compared to baseline (pre-randomization value); K-W=Kruskal-Wallis test; Dunn Test p-value, Double Internalizing vs Double Non-Internalizing; *Dunn Test p-value, Double Non-Internalizing vs Incompletely Internalizing;§Dunn Test p-value, Double Internalizing vs Incompletely Internalizing

[0064] FIG. 17 shows NT -proBNP, Metoprolol Arm. *WSR=Wilcoxon signed rank test compared to baseline (pre-randomization value); K-W=Kruskal-Wallis test; Dunn Test p- value, Double Internalizing vs Double Non-Internalizing; *Dunn Test p-value, Double Non- Internalizing vs Incompletely Internalizing;§Dunn Test p-value, Double Internalizing vs Incompletely Internalizing

[0065] FIG. 18 shows Norepinephrine (NE, pg / ml), Bucindolol Arm. *WSR=Wilcoxon signed rank test compared to baseline (pre-randomization value); K-W=Kruskal-Wallis test; Dunn Test p-value, Double Internalizing vs Double Non-Internalizing; *Dunn Test p-value, Double Non-Internalizing vs Incompletely Internalizing;§Dunn Test p-value, Double Internalizing vs Incompletely Internalizing

[0066] FIG. 19 shows Norepinephrine (NE, pg / m)l, Metoprolol Arm. *WSR=Wilcoxon signed rank test compared to baseline (pre-randomization value); K-W=Kruskal-Wallis test; Dunn Test p-value, Double Internalizing vs Double Non-Internalizing; *Dunn Test p-value, Double Non-Internalizing vs Incompletely Internalizing;§Dunn Test p-value, Double Internalizing vs Incompletely Internalizing

[0067] FIG. 20 shows Target Expansion and Treatment Effect enhancement by haplotype (diplotypes) selection, Treatment Effects in % ((1-Hazard Ratio) x 100). The Cohort is the BEST Adrenergic Receptor Polymorphism Substudy with LVEFs >0.20 (mean+SD 0.273+0.046). MDNI=Maximum Double Non-internalizing diplotypes (2 copies of ADRB1 AND 2 copies of ADRB2 non-internalizing haplotypes). *P value (paired t) <0.01 vs. Columnl; tp <0.01 vs. Column 2; *P<0.05 vs. Column 1;§P value <0.05 vs. Col 2 "P value <0.01 vs. Col5;#P <0.05 vs. Column 5; '][P<0.05 vs. Col 6;AP<0.05 vs. Column 7; 'Either Gly or Arg389 AND Ser49 diplotypes.

[0068] FIG. 21 shows Target Expansion and Treatment Effect enhancement by haplotype (diplotypes) selection, Treatment Effects in % ((1-Hazard Ratio) x 100). The Cohort is the BEST Adrenergic Receptor Polymorphism Substudy with All LVEFs mean+SD 0.236+0.071). MDNI=Maximum Double Non-internalizing diplotypes (2 copies of ADRB1 AND 2 copies of ADRB2 non-internalizing haplotypes). *P value <0.01 vs. Column 1;+P <0.01 vs. Column 2; *P<0.05 vs. Column 1;§P value <0.05 vs. Col 2 "P value <0.01 vs. Column 5; ^P <0.01 vs. Column 6;AP<0.01 vs. Column 7;<either Gly or Arg389 AND Ser49 diplotypes.

[0069] FIG. 22 shows Ratio of pERKl,2 / total ERK1,2 densitometry normalized to vehicle control. *As described in Figures 1 and 23; ^5 minute incubation; *pERKl,2 / total ERK normalized to vehicle = 100%; NDC=nonischemic dilated cardiomyopathy; RCM=Restrictive cardiomyopathy (hemochromatosis); Al = Aortic Insufficiency.

[0070] FIG. 23 shows Forest plot of ADRB1, ADRB2 haplotypes (Column 1) and event rates (Column 5) in patients with LVEFs >0.20 randomized to placebo in the BEST Adrenergic Receptor Polymorphisms substudy. The ADRB1, ADRB2 haplotypes are displayed by "Double Internalizing" combinations in Tier 1 (>3DI, 3 or 4 copies of ADRB1 Arg389Gly49 AND ADRB2 Gln27Argl6; "Incompletely Internalizing" in Tier 2 (211, 2 total copies of internalizing haplotypes, either 1 copy of ADRB1 AND ADRB2, or 2 copies on one of them); and Tier 3, "Double Non-internalizing" haplotype combinations in Tier 3 (>3DNI, 0-1 total copies of Arg389Gly49 OR ADRB2 Gln27Argl6 haplotypes). The copy number in Column 1 designates the number of the specific haplotypes in the diplotype: Haplotype combinations in Column 1 follow "AND" logic, meaning both the ADRB1 and ADRB2 haplotypes must be present in the form designated. The counterpart, comparison haplotypes used to generate ORs follow OR logic, i.e. if either the ADRB1 or the ADRB2 haplotype doesn't match the cognate Column 1 haplotype then the pair becomes a member of the counterpart group. Column 5 lists the event rate and [N] in the paired haplotypes in column 1, followed by the event rate and [N] in the counterpart, comparator group. CVM=Cardio vascular Mortality; AF / AFL / ACM=Atrial fibrillation, Atrial Flutter or All-Cause Mortality; CVM / HFH=CVM or Heart Failure hospitalization; HFH=Heart Failure Hospitalization; ACM / Tx=ACM or Cardiac Transplantation; CVH=Cardiovascular Hospitalization; ACM=A11-Cause Mortality.

[0071] FIG. 24 shows BEST substudy Patients with LVEFs >0.20 randomized to bucindolol in the BEST Adrenergic Receptor Polymorphisms substudy. The forest plot is constructed as for FIG. 23.

[0072] FIG. 25 shows BEST substudy patients with LVEFs >0.20, forest plot of bucindolol vs. placebo treatment effect Odds Ratios (ORs) by haplotype, set-up otherwise as in Figures 1 and 2.

[0073] FIG. 26 shows Odds Ratios in placebo treated subjects from the BEST Adrenergic Receptor Polymorphisms substudy by designated ADRB 1 and ADRB2 haplotype combinations, with ADRB1 Arg389Gly49 held constant and decreasing copies of Gln27Argl6 vs. all other Gln27Argl6 combinations, for 7 clinical endpoints (All LVEF cohort, unadjusted analysis, all races).

[0074] FIG. 27 shows Odds Ratios in bucindolol treated subjects from the BEST Adrenergic Receptor Polymorphism substudy by designated ADRB1 and ADRB2 haplotype combinations with ADRB1 Arg389Gly49 held constant and decreasing copies of Gln27Argl6 vs. all other Gln27Argl6 combinations, for 7 clinical endpoints (All LVEF cohort, unadjusted analysis, all races).

[0075] FIG. 28 shows Odds Ratios for bucindolol vs. placebo treatment effects from the BEST Adrenergic Receptor Polymorphisms substudy by designated ADRB1 and ADRB2 haplotype combinations with ADRB1 Arg389Gly49 held constant and decreasing copies of Gln27Argl6 vs. all other Gln27Argl6 combinations, for 7 clinical endpoints (All LVEF cohort, unadjusted analysis, all races).

[0076] FIG. 29 shows Odds Ratios in placebo treated subjects effects from the BEST Adrenergic Receptor Polymorphisms substudy by designated ADRB1 and ADRB2 haplotype combinations, with ADRB2 Gln27Argl6 held constant and decreasing copies of Arg389Gly49 vs. all other Arg389Gly49 combinations, for 7 clinical endpoints (All LVEF cohort, unadjusted analysis, all races).

[0077] FIG. 30 shows Odds Ratios in bucindolol treated subjects effects from the BEST Adrenergic Receptor Polymorphisms substudy by designated ADRB1 and ADRB2 haplotype combinations, with ADRB2 Gln27Argl6 held constant and decreasing copies of Arg389Gly49vs. all other Arg389Gly49 combinations, for 7 clinical endpoints (All LVEF cohort, unadjusted analysis, all races).

[0078] FIG. 31 shows Odds Ratios for bucindolol vs. placebo treatment effects from the BEST Adrenergic Receptor Polymorphisms substudy by designated ADRB1 and ADRB2 haplotype combinations, with ADRB2 Gln27Argl6 held constant and decreasing copies of Arg389Gly49 vs. all other Arg389Gly49 combinations, for 7 clinical endpoints (All LVEF cohort, unadjusted analysis, all races).

[0079] FIG. 32 shows time to first event curves in placebo treated BEST substudy All LVEF cohort patients with >1 copy (2-4 total copies) of internalizing haplotypes for both ADRB1 AND ADRB2 receptors “Double Internalizing”, DI), vs. all other b-AR receptor haplotypes (“Not Double Internalizing”, NDI). A. All-cause Mortality or Cardiac Transplantation (ACM / Tx); B. Heart Failure Hospitalization (HFH); C. Cardiovascular Mortality or Heart Failure Hospitalization (CVM / HFH); D. Cardiovascular Hospitalization (CVH). Double internalizing receptor haplotypes are associated with improved outcomes in all endpoints.

[0080] FIG. 33 shows time to first event curves in bucindolol treated BEST substudy All LVEF cohort patients with >1 copy (2-4 total copies) of internalizing haplotypes for both ADRB1 AND ADRB2 receptors (“Double Internalizing”, DI), vs. all other b-AR receptor diplotypes (“Not Double Internalizing”, NDI). A. All-cause Mortality or Cardiac Transplantation (ACM / Tx); B. Heart Failure Hospitalization (HFH); C. Cardiovascular Mortality or Heart Failure Hospitalization (CVM / HFH); D. Cardiovascular Hospitalization (CVH). Bucindolol is equally effective in both types of receptor internalization, presumably by internalizing relatively internalization-resistant haplotypes.

[0081] FIG. 34 shows hazard ratios (95% CI) by genotype and haplotype copy number, BEST adrenergic receptor polymorphism substudy (N=1040 in the All LVEF cohort). B= Bucindolol, P=Placebo).

[0082] FIG. 35 shows placebo treatment forest plot, Black / non-Black subjects by internalization tier, All LVEF BEST substudy cohort: Tier 1, >3DI, 3-4 total copies of ADRB1 Arg389 AND ADRB2 Gln27Argl6 (internalizing) haplotypes; Tier 2, 211, 2 total copies of internalizing haplotypes, either 1 copy of ADRB1 AND ADRB2, or 2 copies on one of them); Tier 3, >3DNI ,0-1 total copies ADRB1 OR ADRB2 internalizing haplotypes.

[0083] FIG. 36 shows BEST Adrenergic Receptor Polymorphisms substudy, Treatment Effect % ((1-hazard ratio) x 100) expressed as fold difference vs. All Genotypes / haplotypes subjects (Column / bar 1): Column / bar 2, subjects with an ADRB1 Arg389Arg genotype; (Column / bar 3), ADRB1 Arg389Ser49 diplotype (Dplo; Column / bar 4), Gly389Ser49 diplotype; Column / bar 5, ADRB2 non-internalizing diplotypes; Column / bar 6, ADRB1 AND ADRB2 non-internalizing diplotypes (MDNI); Column / bar 7, ADRB1 Arg389Arg OR ADRB1 AND ADRB2 non-internalizing diplotypes; Column / bar 8, ADRB1 Arg389Ser49 diplotype AND ADRB2 non-internalizing diplotypes.

[0084] FIG. 37 shows ERK1,2 phosphorylation (pERK) and total ERK in preparations of right ventricular trabeculae incubated for 5 or 60 minutes with vehicle (Veh); bucindolol (Buc, le-6M); metoprolol (Met, le-5M); or isoproterenol (Iso, le-6M) (patient 1 in Table 21).

[0085] FIG. 38 shows polymorphisms of the [31 and [32 adrenergic receptors. SNPs producing non-synonymous amino acid changes are indicated in yellow, synonymous changes in red. The beta-2 adrenergic receptor cistronic leader peptide is also depicted. Amino acid changes producing functional effects in receptor proteins are circled, with pharmacologic effects of bucindolol designated.

[0086] FIG. 39A shows a comparison between metoprolol and bucindolol for effects on atrial fibrillation burden (AFB) measured with implanted devices. The GENETIC-AF trial had a 67 patient AFB study using Medtronic ILRs or previously implanted devices, and AFB was markedly reduced by bucindolol, by 55% or 36%, depending on the method used to calculate the difference.19

[0087] FIG. 39B gives GENETIC-AF data for the effects of bucindolol vs. metoprolol on Symptom Burden of AF (SXBAF), the primary endpoint being used in the planned PRECISION- AF trial. IRR = Incidence Rate Ratio (bucindolol arm vs. metoprolol) for symptoms related to atrial fibrillation, in subjects with an LVEF 40% to 55%, within the range of LVEFs of patients who will be enrolled in PRECIS ION- AF.

[0088] FIG. 40 shows a flow chart of the PRECISION-AF trial.

[0089] FIG. 41A-41B shows in vitro studies on ERK 1 / 2. (41 A) Human iPSC-CMs were treated with DMSO or Isoproterenol (ISO, IpM for 15 minutes). Ca2+ transient frequency and time to peak were analyzed using the lonOptix Calcium and Contractility system. (41B)Immunoblot of phosphorylated ERK1 / 2 (pERKl / 2 in top blot) and total ERK1 / 2 (in middle blot) from iPSC-CMs generated from Lamin A / C cardiomyopathy patients with various LMNA mutations. Histogram below blots shows pERKl / 2 activity in iCMs calculated as the ratio of pERKl / 2 over total ERK1 / 2.20

[0090] FIG. 42A shows hazard ratios for haplotypes described herein, newly analyzed data from the 1040 subject BEST trial Adrenergic Receptor Polymorphism DNA Bank substudy21(all subjects, NYHA Class III or IV heart failure) mean LVEF 23.6+7.1 range 5% to 40%).

[0091] FIG. 42B shows the same plot as FIG. 42A, but for 731 subjects with LVEFs from 20% to 40% (mean 27.2+4.6%).

[0092] FIG. 43 shows a flow diagram of the haplotype enhanced treatment effects of bucindolol outlined in FIG. 42A (entire BEST substudy cohort), and the non-internalized (in blue) haplotypes that will be measured in the 400 patient (300 in primary trial and an additional 100 for genotype / haplotype extension) PRECIS ION- AF trial and associated studies designed to expand the indicated population based on targeting a new molecular pharmacogenetic mechanism, biased signaling directed at non-internalizing [3-AR genetic variants.

[0093] FIG. 44 is a haplotype flow chart for internalization-resistant alleles and their impact on treatment effects for the LVEF >20% cohort, displayed identically to FIG. 43 and corresponding to FIG. 5B.

[0094] FIG. 45A-45B. Shows, for the entire LVEF cohort of the BEST DNA substudy, Kaplan-Meier (K-M) curves and Cox adjusted (for the 4 randomization stratification variables) hazard ratios (95% C.I.) plus the P value for the primary endpoint of all-cause mortality or cardiac transplantation (ACM / Tx) in ADRB1 Arg389Arg genotype patients who had no (45A) or 1 or 2 copies of the internalizing, ADRB2 Gln27Argl6 haplotype (45B). Subjects in 45A had ADRB2 haplotypes resistant to internalization.

[0095] FIG. 46A-46B shows the same comparison as in FIG. 45, for the time to AF / AFL / ACM endpoint that will be a high-level secondary endpoint in PRECIS ION-AF.

[0096] FIG. 47A-47B is the same comparison as in FIG. 45, except the subject population has the ADRB1 Gly389 carrier genotype.

[0097] FIG. 48A-48B is the same comparison as in FIG. 46, except that the subject population is ADRB 1 Gly carrier genotype.

[0098] FIG. 49A-49B shows for the LVEF >20% cohort the same comparison as in FIG.45.

[0099] FIG 50A-50B shows for the LVEF >20% cohort the same comparison as in FIG.46.

[0100] FIG 51A-51B. shows for the LVEF >20% cohort the same comparison as in FIG.47.

[0101] FIG 52A-52B shows for the LVEF >20% cohort the same comparison as in FIG.48.DETAILED DESCRIPTION

[0102] The current disclosure generally relates to methods, compositions and kits for treating patients with bucindolol. Pharmacogenomics and / or other criteria allow a clinician or physician to target prophylactic or therapeutic treatments to individuals who will most benefit from the treatment and to avoid treatment of individuals who will experience symptomatic side effects. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics analysis in determining whether to administer bucindolol as well as whether to modify the dosage, regimen, and / or therapeutically effective amounts to be administered so as to attain the effect desired by the treatment. In some aspects, a physician or clinician may alter treatment of the subject by adding an additional therapy.

[0103] Bucindolol can have a selective inhibitory effect on the Arg389 Bl-AR that exceeds standard B-blockers for HF endpoints21 ,37,or reduction in AF burden (AFB), mainly in HFrEF patients.19However, bucindolol’ s “pluridimensionality of efficacy”43,55or “ligand biased signaling”,42has not been exploited for efficacy enhancement. As presented in herein, expansion of the PgT targeted population to include other adrenergic receptor variants, such as ADRB1 Arg or Gly389Ser49 haplotypes and / or non-internalizing ADRB2 haplotypes described herein, increases the eligible target population, in addition to enhancing treatment effect.

[0104] In certain aspects, heart failure patients with non-internalizing [32-AR haplotypes treated with the biased ligand bucindolol, for both the ACM / Tx an AF / AFL / ACM endpointsthe treatment effects are increased to ADRB1 Arg389Arg and Gly389 carriers, as well as to the Arg389Ser49haplotype, as shown herein. Certain aspects are based on the discoveries shown herein that patients with an LVEF of greater than or equal to 20 have an even more pronounced effect on the non-internalizing haplotypes. In further embodiments, the discoveries identified herein demonstrate a consistent difference between the treatment effects of bucindolol in favor of non-internalizing ADRB2 haplotypes (all haplotypes except Gln27Argl6), with attenuation or abolishment of treatment effects when one or two copies of Gln27Argl6 are present.

[0105] Among relatively common cardiovascular disorders only atrial fibrillation (AF) and heart failure (HF) are increasing in prevalence, and at least 25% of AF patients also have HF (AF / HF population). Safe and effective drug therapy in AF / HF is limited, with the few approved agents confined to ion channel inhibitors that have only modest efficacy and significant adverse event profiles. Ideal therapy for AF / HF logically consists of a treatment that would improve both AF and HF, and current drug therapy is directed only at AF prevention. The pathophysiology of AF and HF is similar, consisting of adverse neurohumoral and wall stress activated signaling leading to pathologic chamber remodeling in the atria and ventricles, respectively. A central factor in both is a high level of cardiac adrenergic drive that is a major contributor to pathologic remodeling, via signaling through an extensive gene network downstream from the Pi-adrenergic receptor (Pi-AR). The fh-AR gene (ADR J) is highly polymorphic, with two nonsynonymous SNPs dictating amino acid differences at positions 49 (Ser->Gly, ADRB1 Ser49Gly) and 389 (Arg->Gly, ADRB1 Arg389Gly) that respectively affect receptor internalization and functional coupling. Bucindolol was developed as a 4th generation P-blocker that uniquely affects the major allele protein product of the position 389 polymorphism, and data contained in this application demonstrate that bucindolol also uniquely interacts with the receptor products of position 49 as well as ADRB2 polymorphisms. In HF patients the pharmacogenetic targeting of Arg389 fH-ARs (by selecting patients with an ADRB1 Arg389Arg genotype), bucindolol produces better AF prevention than 2nd or 3rd generation fl-blockers, and is at least as efficacious as ion channel inhibitors.19,22Pharmacogenetically targeted bucindolol also produces enhanced favorable clinical effects in patients with HF and reduced ejection fraction (HFrEF), and uniquely among fl-blockers appears to be effective in the lower ranges of preserved ejection fraction HF (HFpEF).

[0106] Thus far, development of bucindolol has been directed at only the AD RBI Arg3S9Gly polymorphism, based on ADRB1 genotype results from a large (N=1040)adrenergic receptor (AR) polymorphism substudy conducted in an NIH sponsored HF clinical trial, and one Phase 2 AF prevention trial conducted in HF patients homozygous for ADRB1 Arg389. However, aspects herein show clinical efficacy can be further improved by targeting ADRB1 or ADRB2 haplotypes. Aspects herein showing the behavior of these haplotypes suggest that treating patients with 2 copies of the Gly389Ser49 haplotype increases the bucindolol HFrEF (entire cohort of the BEST substudy) target population by 19%, from 47.4% to 56.3% of the U.S. population. For subjects with higher LVEFs (range 20% to 40%) closer to what will be investigated in currently planned trials, the LVEF >20% cohort of the substudy was associated with an increase in pharmacogenetically eligible patients (treatment effects >ADRB1 Arg389Arg) of 57%, from 45.2% to 70.9%. This much higher increase in pharmacogenetic eligibility was due to enhancement of treatment effects by incorporating into the target population subjects with non-internalizing variants of the [32- AR, namely all variants other than the ADRB2 Gln27Argl6 haplotype. In addition, treating patients with 2 copies of the Arg or Gly389Ser49 haplotype increases efficacy compared to treating patients with Arg389Arg genotype that contains a non-responsive genotype, Arg389Gly49. These favorable haplotypes include the ADRB1 position 49 non-internalizing Ser allele, where bucindolol as a “biased ligand” has the ability to internalize the encoded non-intemalized receptors. Aspects herein show the bucindolol favorable treatment effects occur by activating an EGFR / MAP kinase / ERKl / 2 cardioprotective pathway in internalized signalosomes, which will be further tested in human models including cardiac myocytes differentiated from induced pluripotent stem cells. Haplotypes can be estimated to approximately 95% accuracy by probabilistic computer modeling, which, in some aspects, is even more accurate where linkage disequilibrium is present such as for the ADRB1 389 / 49 or ADRB2 27 / 16 polymorphisms. Haplotypes can also be inferred from homozygous genotypes or determined at > 99% accuracy by the recent development of "Long-Read" sequencing technology that accurately and efficiently measures single allele sequences. Aspects herein are interfaced with a 400 patient Phase 3 clinical trial that is designed to generate the final evidence required for FDA approval of bucindolol to prevent AF in HF patents. Pi- and2-AR haplotypes will be measured in the 300 patient efficacy cohort of the trial, as well as in an additional 100 patient supplemental cohort consisting of ADRB1 Gly 389 genotypes not currently eligible for treatment but harboring ADRB1 Gly389Ser49 and ADRB2 haplotypes. The trial utilizes a novel endpoint developed in collaboration with FDA's Division of Clinical Outcomes Assessment that is more efficient than traditional endpoints used in AF trials. As part of this trial a 40 patient substudy will beconducted at CU-AMC to assess left atrial (LA) and left ventricular (LV) reverse remodeling as well as ventricular gene expression differences between bucindolol vs. control (metoprolol succinate) treatment, and their relationship to haplotypes and to each other. This work will be supplemented by gene expression measurements in the same chambers in explanted human hearts.

[0107] Certain aspects concern methods and compositions involving bucindolol (2-(3-(l- ( 1 H-iiulol-3-y l)-2-mcthy lpropan-2-y lamino)-2-hydroxypropoxy)bcnzonitri Ic), which is understood to include bucindolol HC1, unless specifically excluded.

[0108] In some aspects, methods and compositions concern bucindolol, substantially free of its R-stereoisomer. A composition is "substantially free" of R-bucindolol if it includes a mixture of S -bucindolol and (optinally) R-bucindolol wherein the weight of R-bucinolol, if present, is no more than about 20% of the total weight of S -bucindolol and R-bucindolol in the composition. In some aspects, the composition may contain no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.0 % or any range derivable therein by weight of R-bucindolol relative to the total weight of S-bucindolol and R-bucindolol in the composition. In some particular aspects, the composition that is substantially free of R-bucindolol contains no more than about 20% by weight of R-bucindolol relative to the total weight of S-bucindolol and R-bucindolol in the composition. In more particular aspects, the composition contains no more than about 10% by weight of R-bucindolol relative to the total weight of S-bucindolol and R-bucindolol in the composition. In more particular aspects, the inventive composition contains no more than about 10% of R-bucindolol relative to the total weight of S-bucindolol and R-bucindolol in the composition. In even more particular aspects, the inventive composition contains no more than about 1% of R-bucindolol relative to the total weight of S-bucindolol and R- bucindolol in the composition. Additional compositions and methods involving (S)-bucindolol are provided in U.S. Patents 9,446,023, and 9,763,916, which are hereby incorporated by reference.

[0109] Administration of the P-blocker may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, intratracheal, intravesicle, intraocular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further detailson techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.). In certain aspects bucindolol is formulated for oral administration.

[0110] Where clinical applications are contemplated, pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

[0111] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate bucindolol or other active ingredients.

[0112] In some aspects, a patient is treated with a combination therapy that include bucindolol and another pharmaceutical agent.

[0113] In other aspects, the composition may include a nitric oxide (NO) enhancing agent. Examples of NO enhancing agents are well known to those of ordinary skill in the art. Examples of such agents include a RAS inhibitor, a statin, a PDE5 inhibitor, a NO-conjugated drug, or a diazeniumdiolate. Non-limiting examples of RAS inhibitors include captopril, cilazapril, enalapril, fosinopril, lisinopril, quinapril, ramapril, zofenopril, candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, tehnisartan, and valsartan, or a pharmaceutically acceptable salt thereof. Non-limiting examples of statins include atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin calcium, and simvastatin. Non-limiting examples of NO- conjugated drugs include S-NO-glutathione, NO-naproxen, NO-aspirin, NO-ibuprofen, NO- Diclofenac, NO-Flurbiprofen, NO-Ketoprofen, NO-releasing compound-7, NO-releasing compound-5, NO-releasing compound- 12, or NO-releasing compound- 18. Other examples ofNO enhancing agents include L-arginine, arginine alpha-ketoglutarate, GE A 3175, sodium nitroprusside, glyceryl trinitrate, S-nitroso-N-acetyl-penicillamine, nitroglycerin, and diethylamine NONOate. Information concerning NO generating compounds for treating hypertension and atherosclerosis can be found in U.S. Patent 7,396,829, 7,348,319, 7,155,284, 7,052,695, 6,358,536, and 5,208,233, each of which is herein specifically incorporated by reference. Information regarding nebivolol as an NO-enhancing agent can be found in U.S. Patent 7,138,430, herein specifically incorporated by reference.

[0114] Administration of the pharmaceutical compositions set forth herein may be by any method known to those of ordinary skill in the art. Examples include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, intratracheal, intravesicular, intraocular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. Further details on techniques for formulation and administration may be found in the specification below.

[0115] In some aspects, the method further includes contacting the patient with a medical device that includes bucindolol. For example, the medical device may include a coating that includes bucindolol, a matrix that includes bucindolol, or a reservoir that includes a therapeutic composition as set forth above. The device may be inserted into the patient temporarily or implanted in the patient or placed on a body surface of the patient. Examples of such body surfaces include skin surfaces or mucosal surfaces.

[0116] The medical device may be any medical device known to those of ordinary skill in the art. Non-limiting examples of such medical devices include a stent, a graft, a heart valve, a filter, a catheter, a coil, a mesh repair material, a plate, a rod, a screw, or a suture. Aspects also concern medical devices that include a coating, a matrix, or a chamber, wherein the coating, matrix, or chamber includes bucindolol. Non-limiting examples of such medical devices include a stent, a graft, a heart valve, a filter, a catheter, a coil, a mesh repair material, a plate, a rod, a screw, and a suture. An example of a type of filter is an inferior vena caval filter. An example of a type of catheter is a drug infusion catheter. An example of a type of coil is an embolic coil.Detection of polymorphisms

[0117] The presence of the polymorphism can be determined from the sequence of the gene or by using specific characteristics of the polymorphism, e.g., restriction enzyme recognitionsite, hybridization, etc. As a result, a variety of different methodologies can be employed for the purpose of detecting polymorphisms in genes. Alternatively, the protein gene product can be evaluated to determine the patient’s genotype.Nucleic Acids

[0118] Certain aspects concern various nucleic acids, including amplification primers, oligonucleotide probes, and other nucleic acid elements involved in the analysis of genomic DNA. In certain aspects, a nucleic acid comprises a wild-type, a mutant, or a polymorphic nucleic acid.

[0119] In some aspects, nucleic acids used in aspects comprise or are complementary to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1165, 1200, 1300, 1400, 1500, or more contiguous nucleotides, or any range derivable therein, of the human PiAR gene or the human P2AR gene. One of skill in the art knows how to design and use primers and probes for hybridization and amplification of a sequence in the human PiAR or human P2AR gene. In some aspects, the sequence is the PiAR or P2AR coding sequence (or its complement) or it is based on the PiAR or P2AR transcript, such as a cDNA of this sequence.

[0120] These definitions generally refer to a single-stranded molecule, but in specific aspects will also encompass an additional strand that is partially, substantially or fully complementary to the single- stranded molecule. Thus, a nucleic acid may encompass a doublestranded molecule or a triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a molecule. As used herein, a single stranded nucleic acid may be denoted by the prefix "ss", a double stranded nucleic acid by the prefix "ds", and a triple stranded nucleic acid by the prefix "ts."Preparation of Nucleic Acids

[0121] A nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production or biological production. Non-limiting examples of a synthetic nucleic acid (e.g., a synthetic oligonucleotide), include a nucleic acid made by in vitro chemical synthesis using phospho triester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in European Patent 266,032, incorporated herein by reference, or via deoxy nucleoside H-phosphonate intermediates as described by Froehler et al., 1986 and U.S. Patent 5,705,629, each incorporated herein by reference. In the methods of the present invention, one or more oligonucleotides may be used. In certain aspects amplification oligonucleotides can be designed on either side or overlapping with the boundaries of the insertion site. In a further aspect an oligonucleotide specific for the sequence at 894, whether a G or a T, can be designed. These oligonucleotides can varying in length from 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, nucleotides or more, including all values and ranges there between. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.

[0122] A non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCR™ (see for example, U.S. Patent 4,683,202 and U.S. Patent 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U.S. Patent 5,645,897, incorporated herein by reference. A non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (z.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria (see for example, Sambrook et al. 2001, incorporated herein by reference).Purification of Nucleic Acids

[0123] A nucleic acid may be purified on polyacrylamide gels, cesium chloride centrifugation gradients, chromatography columns or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al., 2001, incorporated herein by reference).

[0124] Certain aspects concern a nucleic acid that is an isolated nucleic acid. As used herein, the term "isolated nucleic acid" refers to a nucleic acid molecule (e.g. , an RNA or DNA molecule) that has been isolated free of, or is otherwise free of, the bulk of the total genomicand transcribed nucleic acids of one or more cells. In certain aspects, "isolated nucleic acid" refers to a nucleic acid that has been isolated free of, or is otherwise free of, bulk of cellular components or in vitro reaction components such as for example, macromolecules such as lipids or proteins, small biological molecules, and the like.Nucleic Acid Complements

[0125] Aspects also encompass a nucleic acid that is complementary to a nucleic acid. A nucleic acid is "complement(s)" or is "complementary" to another nucleic acid when it is capable of base-pairing with another nucleic acid according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules. As used herein "another nucleic acid" may refer to a separate molecule or a spatial separated sequence of the same molecule. In certain aspects, a complement is a hybridization probe or amplification primer for the detection of a nucleic acid polymorphism.

[0126] As used herein, the term "complementary" or "complement" also refers to a nucleic acid comprising a sequence of consecutive nucleobases or semiconsecutive nucleobases (e.g., one or more nucleobase moieties are not present in the molecule) capable of hybridizing to another nucleic acid strand or duplex even if less than all the nucleobases do not base pair with a counterpart nucleobase. However, in some diagnostic or detection aspects, completely complementary nucleic acids are used.Nucleic Acid Detection and Evaluation

[0127] Genotyping can be performed using methods described in Small et al. (2002), which is incorporated herein by reference. It will be understood by the skilled artisan that other standard techniques are available for genotyping and any technique may be used with the aspects described herein. General methods of nucleic acid detection methods are provided below.

[0128] In some aspects, genotyping involves isolating from the patient a nucleic acid mixture comprising both copies of the CYP2D6 or PiAR gene, or a fragment thereof, and determining the nucleotide sequence of one or more polymorphisms. In some aspects, this involves determining the sequence based on the transcripts produced from both copies of the gene. Other polymorphisms, such as single nucleotide polymorphisms can be linked to and indicative of the polymorphism at positions described herein. Consequently, in some aspects apolymorphism in linkage disequilibrium (LED or LD) with a polymorphism may be used to determine the sequence at a different position.

[0129] Those in the art will readily recognize that nucleic acid molecules may be doublestranded molecules and that reference to a particular site on one strand refers, as well, to the corresponding site on a complementary strand. Thus, in defining a polymorphic site, reference to a sequence including an adenine, a thymine (uridine), a cytosine, or a guanine at a particular site on one strand of a nucleic acid molecule is also intended to include the thymine (uridine), adenine, guanine, or cytosine (respectively) at the corresponding site on a complementary strand of a nucleic acid molecule. Thus, reference may be made to either strand and still comprise the same polymorphic site and an oligonucleotide may be designed to hybridize to either strand.

[0130] Typically, the nucleic acid mixture is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample using standard techniques such as disclosed in Jones (1963) which is hereby incorporated by reference. Suitable tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin, and hair. The nucleic acid mixture may be comprised of genomic DNA or cardiac RNA

[0131] In the genotyping methods used in aspects, the identity of a polymorphic site may be determined by amplifying a target region containing the polymorphic site directly from one or both copies of the gene present in the individual and the sequence of the amplified region(s) determined by conventional methods or evaluated directly.

[0132] The target region(s) may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent 4,965,188), ligase chain reaction (LCR) (Barany et al., 1991; W090 / 01069), and oligonucleotide ligation assay (OLA) (Landegren et al., 1988). Oligonucleotides useful as primers or probes in such methods should specifically hybridize to a region of the nucleic acid that contains or is adjacent to the polymorphic site. Typically, the oligonucleotides are between 10 and 35 nucleotides in length and preferably, between 15 and 30 nucleotides in length. Most preferably, the oligonucleotides are 20 to 25 nucleotides long. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.

[0133] Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent 5,130,238; EP 329,822; U.S. Patent 5,169,766, W089 / 06700) and isothermal methods (Walker et al., 1992).

[0134] A polymorphism in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art. Typically, allele- specific oligonucleotides are utilized in performing such methods. The allele- specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant. In some aspects, more than one polymorphic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs.

[0135] Hybridization of an allele- specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele- specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis. Solidsupports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads. The solid support may be treated, coated or derivatized to facilitate the immobilization of the allele- specific oligonucleotide or target nucleic acid.

[0136] The identity of polymorphisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al., 1985; Meyers et al., 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, 1991). Alternatively, variant alleles can be identified by single strand conformation polymorphism (SSCP) analysis (Orita et al., 1989; Humphries et al., 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989).

[0137] A polymerase-mediated primer extension method may also be used to identify the polymorphism(s). Several such methods have been described in the patent and scientificliterature. Extended primers containing a polymorphism may be detected by mass spectrometry as described in U.S. Pat. No. 5,605,798. Another primer extension method is allele- specific PCR (Ruano et al., 1989; Ruano et al., 1991; WO 93 / 22456; Turki et al., 1995).

[0138] Polymorphic variation in genes can also be detected using differential digestion of DNA by certain restriction enzymes (Small et al., 2002) or by any other method that identifies the sequence of the polymorphic position in the gene.

[0139] In a specific example, amplification and sequencing of nucleic acids from biological samples of the set of biological samples includes: solid-phase PCR involving bridge amplification of DNA fragments of the biological samples on a substrate with oligo adapters, wherein amplification involves primers having a forward index sequence (e.g., corresponding to an Illumina forward index for MiSeq / NextSeq / HiSeq platforms) or a reverse index sequence (e.g., corresponding to an Illumina reverse index for MiSeq / NextSeq / HiSeq platforms), a forward barcode sequence or a reverse barcode sequence, a transposase sequence (e.g., corresponding to a transposase binding site for MiSeq / NextSeq / HiSeq platforms), a linker (e.g., a zero, one, or two-base fragment configured to reduce homogeneity and improve sequence results), an additional random base, and a sequence for targeting a specific target region (e.g., 16S region, 18S region, ITS region). Amplification and sequencing can further be performed on any suitable amplicon, as indicated throughout the disclosure. In the specific example, sequencing comprises Illumina sequencing (e.g., with a HiSeq platform, with a MiSeq platform, with a NextSeq platform, etc.) using a sequencing-by-synthesis technique. Additionally or alternatively, any other suitable next generation sequencing technology (e.g., PacBio platform, MinlON platform, Oxford Nanopore platform, etc.) can be used. Additionally or alternatively, any other suitable sequencing platform or method can be used (e.g., a Roche 454 Life Sciences platform, a Life Technologies SOLiD platform, etc.). In examples, sequencing can include deep sequencing to quantify the number of copies of a particular sequence in a sample and then also be used to determine the relative abundance of different sequences in a sample. Deep sequencing refers to highly redundant sequencing of a nucleic acid sequence, for example such that the original number of copies of a sequence in a sample can be determined or estimated. The redundancy (i.e., depth) of the sequencing is determined by the length of the sequence to be determined (X), the number of sequencing reads (N), and the average read length (L). The redundancy is then N. times. L / X. The sequencing depth can be, or be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 300, 500, 500, 700, 1000, 2000, 3000, 4000, 5000 or more.Sequencing

[0140] Aspects of the disclosure may include sequencing nucleic acids to determine whether a gene has one or more polymorphisms.. Sequencing technology that has been available for several years includes massively parallel signature sequencing (MPSS) developed in the 1990s at Lynx Therapeutics, the polony sequencing method developed by George M. Church at Harvard in 2005, and 454 pyrosequencing developed in 2006 by 454 Life Sciences (acquired by Roche Diagnostics). In some aspects, they are superseded by new technologies, such as those disclosed below. The first next-generation sequencing technologies included Illumina (Solexa), SOLiD, Ion Torrent semiconductor, DNA nanoball, and Heliscope single molecule sequencing methods. The third generation of sequencing technologies includes single molecule real time (SMRT), Nanopore real time long read, and Illumina and 10X Genomics synthetic long read sequencing technologies. Long read sequencing may comprise methods described in Pendelton et al. (Nat Methods. 2015 Aug;12(8):780-6. Doi: 10.1038 / nmeth.3454.)

[0141] The term "polymorphism", as used herein, refers to a difference in the nucleotide or amino acid sequence of a given nucleotide or amino acid region as compared to a nucleotide or amino acid sequence in the corresponding region of another individual of the same species. Preferably, the species is human. A polymorphism is generally defined in relation to a "reference" sequence. In the subject application, "reference" sequence and "wild type" sequence are used interchangeably. Nucleotide polymorphisms include single nucleotide differences, differences in sequence of more than one nucleotide, and single or multiple nucleotide insertions, inversions, substitutions, and deletions. Amino acid polymorphisms include single amino acid differences, differences in sequence of more than one amino acid, and single or multiple amino acid insertions, substitutions, and deletions.

[0142] A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definitionalso includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term biological sample encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples. In one aspect, the sample is collected by the individual. For example, an individual can collect a swap of tissue from the inside of the cheek for use as a nucleic acid sample. As known in the art, many types of samples can be used for the extraction of nucleic acids.

[0143] In another aspect, a further step is added wherein a portion of a gene is amplified prior to the identifying step. In another aspect, the identifying is performed by a method selected from the group consisting of a hybridization assay, a sequencing assay, a microsequencing assay, a MALDI-TOF assay, and an allele- specific amplification assay. In a further aspect, the identifying is performed by an antibody-based assay.

[0144] Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to individuals who will most benefit from the treatment and to avoid treatment of individuals who will experience symptomatic side effects. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer bucindolol as well as tailoring the dosage, regimen, and / or therapeutically effective amounts to be administered so as to attain the effect desired by treatment with the modulator.Administration of Therapeutic Compositions

[0145] Aspects herein relate to administration of therapeutic compositions, including bucindolol. In some aspects, a therapeutic composition comprising bucindolol is administered to a patient. Aspects also relate to methods comprising administering an effective amount of a beta-blocker, such as bucindolol. In certain aspects, one or more additional therapeutics are administered to the patient. The additional therapeutics may further treat a disease, disorder, or symptoms disclosed herein.

[0146] In some aspects, the first therapeutic composition and the second therapeutic composition are administered substantially simultaneously. In some aspects, the first therapeutic composition and the second therapeutic composition are administered sequentially.In some aspects, the first therapeutic composition, the second therapeutic composition, and a third therapeutic composition are administered sequentially. In some aspects, the first therapeutic composition is administered before administering the second therapeutic composition. In some aspects, the first therapeutic composition is administered after administering the second therapeutic composition.

[0147] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0148] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0149] Precise amounts of the therapeutic composition may depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0150] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels). It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0151] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 day, week, month, or year intervals, including all ranges there between.EXAMPLESExample 1: Haplotype Targeting in Heart Failure Using a Biased Ligand P-blockerINTRODUCTION

[0152] Human cardiac myocytes contain 3 types of P-adrenergic receptors (fLARs), with Pi-ARs dominating but fh-ARs also potentially involved in regulation of cardiac function and capable of mediating pathologic effects of chronic adrenergic stimulation in the failing heart.1,2Increased cardiac adrenergic drive signaling through fh-ARs is responsible for an important component of pathologic eccentric remodeling in heart failure (HF) patients, the inhibition of which largely explains the beneficial effects of P-b lockers reduced ejection fraction HF (HFrEF).1,3,4Chronically increased cardiac adrenergic drive has a net myopathic effect on the myocardium including predisposing to arrhythmias,1,4,5certain cellular mechanisms can counter these adverse effects. The most obvious of these are P-AR desensitization and / or downregulation that diminishes adrenergic signaling through the canonical cAMP / PKA pathway.1In addition, for both Pi-6-8and Pi-ARs9 11desensitization also includes signaling through an alternative, EGFR and ERK1 / 2 cardioprotective pathway consisting of receptor phosphorylation, binding to P-arrestins, endocytic internalization and endosome trafficking to the cytosol.7,8In this series of events receptor internalization is a critical step, as inhibitors of endocytic vesicle formation block EGFR transactivation and ERK1,2 phosphorylation.10,12P- adrenergic agonists classically activate this pathway, but with some differences13so can certain P-blocker "biased ligands" that have relatively less efficacy for canonical, G-protein / adenylate cyclase / c AMP-PKA activation.14,15

[0153] In addition to agonist and biased ligand activation of ERK1 / 2 signaling, some P- AR polymorphisms internalize on exposure to agonists to a much greater degree than their counterpart, more commonly expressed variants. Based on work done in the Gly389 variant of human ADRB1 the more prominently internalizing Pi-AR variant is Gly49,16,17and using agonist-induced rapid desensitization and / or downregulation with no change in mRNA abundance as proxies for internalization it appears that ADRB1 Gly49 behaves similarly wheninvestigated as a haplotype with either Arg or Gly389.18For f -ARs, the more-internalizing variant(s) is less clear, but the ADRB2 Gln27Argl6 haplotype exhibits rapid agonist desensitization and downregulation compared to other haplotypes.19,20The counterpart, lessor non-internalizing variants for Pi- and i-ARs are respectively ADRB1 Ser49 containing genotypes and haplotypes,16 18and ADRB2 non-Gln27Argl6 haplotypes.20

[0154] The ADRB1 49 and the ADRB2 27 and 16 polymorphisms do not affect signal transduction efficiency or agonist affinity.16 19However, for ADRB1 the Ser49Gly polymorphism is in strong linkage disequilibrium with the nonsynonymous SNP Arg389Gly,21the encoded receptors of which have major effects on signal transduction capacity and agonist affinity.22,23The ADRB2 position 27 and 16 variants are also in tight linkage disequilibrium.21ADRB1 Arg389Gly / Ser49Gly haplotypes can therefore encode receptors with greater or lesser pharmacologic activity that internalize to a greater or lesser extent in the presence of agonists or “biased”24ligands, while the ADRB2 Gln27Argl6 vs. other encoded haplotypes have the potential for more vs. less internalization.

[0155] In the MAP-MI study21the inventors recently reported that the ADRB1 Arg389Gly49 haplotype, which encodes a high functioning, easily internalized f -AR, is associated with a reduction in incident ventricular fibrillation in acute myocardial infarction, possibly due to EGFR / ERK1 / 2 cardioprotective pathway signaling. An earlier study had found that the ADRB1 Gly49 polymorphism was associated with improved survival in dilated cardiomyopathy patients, also suggesting a myocardial protective mechanism.25In MAP-MI there was also evidence of a cardioprotective signal in subjects with the internalizing ADRB2 Gln27Argl6 haplotype.21Since HFrEF human ventricles contain 35-40% fh-ARs that are coupled to inotropic and pro-arrhythmic pathways and when overexpressed in model systems to a cardiomyopathy,1,2there is also a rationale for fh-AR internalization affecting outcomes in HFrEF.

[0156] Here the inventors test the hypothesis that internalization properties of ADRB1 and ADRB2 common genetic haplotype variants affect clinical outcomes in HFrEF patients treated with a biased ligand P-b locker (bucindolol), compared to placebo or a nonbiased P-b locker (metoprolol succinate). The 2 clinical trial cohorts investigated23,26were haplotyped for ADRB1 and ADRB2 variants, and one of them (the BEST Trial Adrenergic Receptor Polymorphism substudy23,27) included populations with substantial racial heterogeneity that affects haplotypefrequency. The presented data support the utility of pharmacogenetic targeting of protein haplotypes,28something that has long held promise29,30but has been difficult to implement.RESULTSBEST Adrenergic Receptor Polymorphism Substudy

[0157] This is a 1040 patient pharmacogenomics substudy23of the BEST trial27that investigated advanced HFrEF patients (NYHA Class III-IV HF, LVEFs <0.35) with elevated plasma norepinephrine (NE) levels (FIG. 7) treated with bucindolol or placebo. Eligible subjects gave written informed consent for the parent trial27and as an option for peripheral blood DNA extraction and genotyping in the pharmacogenomics substudy.23Further trial details are in STAR Methods.Allele, Genotype and Haplotype Frequencies; Linkage Disequilibrium

[0158] A description of the formatting and terminology used for genotypes, haplotypes and diplotypes is in the first paragraph of Supplemental Materials. Allele frequencies of the ADRB1 and ADRB2 polymorphisms by race are in FIG. 8, and are similar to those previously reported for European- vs. African-Ancestry in the BEST substudy.31Allele frequencies of all 4 ADRB1 or ADRB2 polymorphisms investigated differ by Black vs. non-Black race, which leads to higher minor allele frequencies in Blacks for ADR J 389Gly and 49Gly, and ADRB2 16Arg. ADRB2 27Glu has a lower frequency in Blacks. Genotypes and theoretical vs. actual identified diplotypes are in FIG. 9. For both ADRB1 and ADRB2 there are 10 theoretical diplotypes (4 homozygotes, 4 single hetero zygotes, 2 double heterozygotes), but only 6 were identified; diplotypes with ADRB1 Gly389Gly49 or ADRB2 Glu27Argl6 haplotypes are missing. Identified diplotypes in each gene consist of 3 homozygotes, 2 single heterozygotes, and 1 double heterozygote. There is strong linkage disequilibrium (LD) between ADRB1 positions 389 and 49 polymorphisms, as well as among ADRB2 positions 27, 16 and 164 (FIG. 10). The absence of the Arg389Ser49 / Gly389Gly49 and Gln27Glyl6 / Glu27Argl6 haplotypes was confirmed by long-read DNA sequencing in 26 ADRB1 and 50 ADRB2 double heterozygotes.

[0159] Diplotypes by Black and non-Black race are in Table 1, with haplotype frequencies in FIG. 11. For ADRB1 the haplotype frequencies differ by race; the ADRB1 Arg389Ser49, non-internalizing homozygote haplotype is 1.76 fold more frequent in non-Blacks (P <0.0001, FIG. 11), with an Arg389 / Ser49 containing (“diplotype carrier”) frequency 1.46 fold higher innon-Blacks (P <0.0001, FIG. 7). Due to higher allele frequency of Gly389, in Blacks the Gly389Ser49 (non-internalizing ) haplotype frequency is 1.71 fold higher than in non-Blacks (P <0.0001, FIG. 11), with diplotype carriers 1.39 fold higher(Table 1, P<0.0001). For the combined Arg and Gly389 / Ser49 containing haplotypes the frequency is 1.13 fold in favor of non-Blacks (FIG. 11, P <0.0001). Blacks have higher frequencies than non-blacks for the Arg389Gly49 ADRB1 internalizing) haplotype, by 1.71 fold )(P <0.0001, FIG. 11), with diplotype carriers 1.64 fold higher (P <0.0001, Table 1). For ADRB2, the Gln27Argl6 (internalizing) haplotype frequency is 1.21 fold higher in Blacks vs. non-Blacks (P=0.004) (FIG. 11), with diplotype carriers 1.19 fold higher in Blacks (P=0.0012) (Table 1). Non-Blacks compared to Blacks have a higher (by 1.15 fold) frequency of non-internalizing haplotypes (all haplotypes not containing Gln27Argl6, "Not Gln27Argl6", P= 0.0012, FIG. 11) as well as a higher diplotype carrier frequency (by 1.46 fold, (P= 0.0012, Table 1).Table 1. ADRB1, ADRB2 diplotype frequencies ( / ) by race, BESTAdrenergic Receptor Polymorphism Substudy.f= Diplotype frequency (Diplotype N / Total N).Baseline Characteristics by Haplotype

[0160] For baseline characteristics by diplotype the main differentiating characteristic is race (FIG. 7), a product of its association with differences in allele and haplotype frequencies (Tables 1 and FIG. 11). For ADRB1, Arg389Ser49 homozygotes have fewer Blacks (P <0.001) than in other diplotypes, while due to the higher allele frequency of Gly389, Gly389Ser49 homozygotes and Arg389Gly49 / Gly389Ser49 heterozygotes have more Blacks (P <0.001, <0.0001 respectively). Other demographic features within ADRB1 diplotypes are well balanced. For ADRB2 the non-internalizing ADRB2 Glu27Glyl6 homozygous diplotype and the Gln27Arl6 / Glu27Glyl6 double heterozygote have fewer Blacks (P <0.001), and the Gln27Argl6 / Gln27Glyl6 single heterozygote has more Blacks (P <0.001). Similar to ADRB1, other demographic features are well balanced among ADRB2 diplotypes (FIG. 11).Clinical Outcomes Data by Haplotype Internalization PotentialOdds Ratios for Clinical Events, Internalizing vs. Non-internalizing Haplotypes

[0161] To test the hypothesis that internalizing ADRB1 and ADRB2 haplotypes affect clinical outcomes, in placebo- and bucindolol-treated subjects in the BEST adrenergic receptor polymorphism substudy unadjusted haplotype odds ratios (ORs) for clinical event rates werecalculated by the number of internalizing haplotypes (ADRB1 Arg389Gly49, ADRB2 Gln28Argl6) per diplotype combination and compared to remaining, non-internalizing haplotypes (see descriptor classification, FIG. 12). In the classification descriptors the “number” is the sum of the ADRB1 + ADRB2 haplotypes in the diplotype, “D” (double) indicates that at least 1 defining haplotype is present in both genes, “I” is internalizing, and “NI is non-internalizing. Figures 1-3 (entire all-LVEF cohort) and Figures 23-25 (cohort restricted to baseline LVEFs of >0.20) are forest plots of the between-haplotypes ORs for 7 clinical endpoints that include the primary endpoints for the BEST substudy (time to all-cause mortality or cardiac transplantation, ACM / Tx)23and GENETIC-AF (time to first atrial fibrillation or flutter, or ACM (AF / AFL / ACM)).26Unadjusted summary statistics for the aggregate endpoint analyses are in FIG. 13, with a covariate adjusted, per-subject endpoint structural equation model (SEM) analysis in FIG. 14. The readout is in first event rates for the entire N=1029 patient BEST substudy cohort in subjects who had complete ADRB1 and ADRB2 genotyping. In Figures 1-3 and 23-25 the ADRB1 and ADRB2 haplotype combinations are organized by 3 sections or tiers based on the total number of internalizing potential ADRB1 and ADRB2 haplotypes, with the tier requirements in the first column. In Tier 1, 3-4 copies of ADRB1 AND ADRB2 internalizing haplotypes is “Double-Internalizing", >3DI); Tier 2 has 2 copies of internalizing haplotypes ("Incompletely Internalizing", 21); and Tier 3 has 0-1 copies of internalizing (3-4 non-internalizing) haplotypes ("Double Non- internalizing", >3DNI)). The sequence of presentation is placebo treated patients (Figures 1 and 23), bucindolol patients (Figures 2 and 24), and bucindolol vs. placebo treatment effects (Figures 3 and 25). ORs in Figures 1 / 23 and 2 / 24 are generated by comparing the haplotype pairs in Column 1 to all other haplotype pairs not defined in Column 1, and treatment effect OR (Figures 3 / 25) are based on event ORs of bucindolol vs. placebo.With Placebo Treatment ADRB1 + ADRB2 Internalizing vs. Non-internalizing Haplotypes are Associated with Lower Clinical Event Rates

[0162] In the All-LVEF cohort the endpoint analysis of placebo treatment (FIG. 1) indicates that subjects within Tier 1 (>3DI / Double Internalizing) have OR point estimates substantially to the left of the line of identity, ranging from 0.29 (0.12, 0.68) for cardiovascular mortality or heart failure hospitalization (CVM / HFH) to 0.48 (0.23, 1.00) for cardiovascular hospitalization (CVH). The mean across all 7 endpoints is 0.39 (0.27, 0.55). In contrast, for the Tier 3, >3DNI / / Double Non-internalizing subjects ORs are to the right of the line of identity, between 1.45 (0.88, 2.39) for CVM and 1.78 (1.22, 2.58) for CVH, mean 1.64 (1.46, 1.84). TheTier 2, 2I / Incompletely Internalizing haplotype subjects in FIG. 1 exhibit positioning of the 7 OR point estimates intermediate between Tiers 1 and 3, and slightly to the left of the identity line with a mean OR of 0.76 (0.66, 0.88). By an aggregate analysis of all 7 endpoints, Tier 1 event rates and their ORs are significantly different from Tier 3 (both P=0.005, FIG. 13), and growth curve analysis of the event rates yields a positive slope of 1.60 (P<0.001). The persubject, SEM analysis (FIG. 20) adjusted for both dependencies between endpoints and direct effects of 12 covariates confirms a progressive increase in the placebo event rate slopes for Tier 1 through Tier 3; all 7 endpoints in both LVEF cohorts have positive individual slopes, and 5 are P <0.05. The slope calculation gives a linear estimate of the fractional change in event rates proceeding from each tier to the next, such that for the BEST substudy primary endpoint of ACM / Tx placebo treated subjects in the All LVEF cohort have an increase in event rate by 5.8% in Tier 2 vs. Tier 1, and another 5.8 % increase in Tier 3 vs Tier 2. Data for placebo treatment for the LVEF >0.20 subjects (FIG. 23 and 14) are similar, but the event rate slope values are numerically higher than in the All-LVEF cohort.Bucindolol Treatment is Minimally or Not Affected by Haplotype Internalizing Potential

[0163] In contrast to placebo treatment, the bucindolol forest plots for the All-LVEF (FIG. 2) and LVEF >0.20 cohorts (Figures 2, 24, 13, 14) exhibit less tendency to segregate with event rates or between haplotype ORs, with nonsignificant event rate growth curve slopes in the aggregate analysis (FIG. 13). or SEM composite endpoints analysis (FIG. 14, 0.10, P=0.22). For the SEM analysis (FIG.14), in contrast to placebo no slope value is statistically significant, i.e. on the covariate adjusted analysis there is no change in event rates for either LVEF cohort moving across the 3 tiers in Figures 2 and 24.Treatment Effects of Bucindolol are Inversely Related to Haplotype Internalizing Potential

[0164] Figures 3 and 25 contain between-treatment group ORs within internalization haplotype tiers, and demonstrate a point estimate pattern commensurate with the within- placebo, between-haplotype ORs in Figures 1 and 23 and the lack of effects by internalization tier in bucindolol subjects in Figures 2 and 24. In both the All-LVEF (FIG. 3) and LVEF >0.20 (FIG. 25) cohorts the 3-4 (>3DI double internalization haplotype Tier 1 ORs, although statistically nonsignificant, are to the right of the line of identity, while in Tier 3 (>3DNI,Double Non-internalizing) the ORs are <1.0 with the majority (5 of 7 in FIG. 3, all 7 in FIG. 25) statistically significant.

[0165] The aggregate unadjusted treatment effect analysis (FIG. 13) indicates a highly significant (P<0.001) negative slope (0.50) for OR decrease across the 3 haplotype tiers in the LVEF >0.20 cohort, and a nonsignificant (P=0.074) negative slope (0.72) in the All LVEF cohort. The adjusted, SEM analysis (FIG. 14) indicates consistent treatment effects across all endpoints in both LVEF cohorts, with negative slopes throughout and all but ACM in the All LVEF cohort being statistically significant. The BEST substudy primary endpoint of ACM / Tx yields a composite reduction in event rates of 12.2 (P=0.044) and 17.4 (P=0.001) absolute % in the All LVEF and LVEF >0.20 cohorts respectively, while the currently favored HF primary endpoint of CVM / HFH yields respective reductions of 24.8 (P=0.005) and 36.6% (P <0.0001) (FIG. 14).ADRB1 and ADRB2 Haplotypes Yield Similar Internalization to N on-internalization Dose- ResponsesPlacebo Treatment

[0166] Figures 26-28 contain All-LVEF cohort data for the internalizing ADRB1 Arg389Gly49 haplotype held constant at 1 or 2 copies and paired with decreasing copies (from 2 to 0) of the ADRB2 internalizing, Gln27 / Argl6 haplotype. ORs are calculated and plotted as for Figures 1-3, representing an internalizing to non-internalizing ADRB2 haplotype doseresponse for the 7 clinical endpoints. In placebo-treated patients (FIG. 26), within each clinical endpoint there is a progressive increase in ORs for all 7 endpoints as the copy number of ADRB2 Gln27Argl6 decreases, from an OR range of 0.15 to 0.47 (mean 0.28+0.12) for 2 copy and 1.03 to 1.59 (mean 1.34+0.19) for 0 copies (P=0.016). For the 7 clinical endpoints, 8 of the 14 ORs in the Gln27Argl6 2 or 1-2 copy groups were statistically significantly (upper bound <1.00) reduced, while none were significantly changed in the remaining 3 categories with fewer copy numbers. The converse, haplotype downtitration of ADR B J Arg389Gly49 in the presence of constant, 1-2 copies of ADRB2 Gln27Argl6 (FIG. 29) yields similar data.Bucindolol Treatment

[0167] In contrast, the internalizing to non-internalizing haplotype dose response is not present in patients treated with bucindolol (Figures 27, 28). In FIG. 27, with downtitration of the internalizing ADRB2 haplotype Gln27Argl6, ORs are on either side and close to the 1.0 line of identity with none statistically significant. The downtitration of the ADRB1 internalizinghaplotype (FIG. 30) reveals a slightly different pattern, with 2 copies of Arg389Gly49 demonstrating a lower OR (ranging from 0 to 0.73, mean 0.32+0.29) than all other copies in all 7 endpoints. Also, for mortality dominant endpoints (CVM, ACM / Tx, ACM), haplotypes that with 0 copies of Arg389Gly49 have ORs consistently >1.0 (range 1.25-1.68, mean 1.52+0.16). These 2 deviations from Gln27Argl6 downtitration are likely due to the respective favorable and relatively unfavorable impacts of the ADRB1 Arg389Arg and Gly389 genotypes on bucindolol effects on HF endpoints.23Bucindolol vs. Placebo Treatment Effects

[0168] Bucindolol vs. placebo treatment effects with downtitration of internalizing haplotypes (Figures 28 and 31) generally indicate an overall progressive decrease in ORs in all endpoints, or the opposite pattern to that for placebo in Figures 26 and 29. The one exception is consistently low ORs for 2 copy Arg389Gly49 in all 7 endpoints, again likely due to the favorable impact of the ADRB1 Arg389Arg genotype on bucindolol treatment effects.23Hazard Ratios for Time to Clinical Events, Internalizing vs. Non-internalizing HaplotypesPlacebo or Bucindolol Treatment in Internalizing vs. Non-internalizing Haplotypes

[0169] Clinical event hazard ratios (HzRs) differ from odds ratios by incorporating the element of time, which can increase detection sensitivity for treatment effect differences. For placebo treated patients in the BEST substudy All LVEF cohort, FIG. 32 contains time to first event curves and HzRs comparing >2 Double Internalizing (>2DI, 1-2 copies of ADRB1 Arg389Glyl6 AND 1-2 copies of ADRB2 Gln27Argl6) to subjects with all other (>2 Not Double Internalizing, >2NI) haplotypes, for 4 of the clinical endpoint ORs plotted in Figures 1-3 and 23-25. In all 4 endpoints placebo-treated subjects with >2DI haplotypes exhibit progressive, favorable separation in the Kaplan-Meier (K-M) curves compared to >2NI haplotypes, with HzRs ranging from 0.62 (0.40, 0.97) to 0.69 (0.48, 1.00). FIG. 33 contains the same comparisons for bucindolol-treated patients in the ALL LVEF cohort, where curve separation does not occur for >2DI vs. >2NIsubjects; HzRs are near 1.0 for all 4 endpoints, and event rates are similar to the >2DI placebo curves in FIG. 32.Bucindolol vs. Placebo Treatment Effects in Maximum Non-internalizing Haplotypes

[0170] As an extreme evaluation of the consequences of non-internalizing ADRB1 and ADRB2 haplotypes on treatment effects, in the LVEF >0.20 cohort bucindolol vs. placebo time to first event curves for “Maximum Double Non-internalizing” (4DNI) ADRB1 and ADRB2diplotypes (2 copies of an ADRB1 Ser49 containing haplotype AND 2 copies of an ADRB2 non-Gln27Argl6 diplotype) are shown in FIG. 4. HzRs (95% C.I.s) range from 0.17 (0.05, 0.56) for ACM / Tx (substudy primary endpoint) to 0.53 (0.33, 0.84) for CVH.ADRBI and ADRB2 Haplotypes Dose Impact on Bucindolol Treatment Effect Hazard Ratios

[0171] Based on data presented in Figures 1-3 and 23-25 as well as in FIG. 13 bucindolol treatment effects appear to be directly related to the presence of non-internalizing ADRBI and ADRB2 haplotypes. FIG. 34 contains data further addressing this issue, by examining the effects of copy numbers of specific haplotypes on the ACM / Tx and AF / AFL / ACM primary endpoint HzRs. Rows E1 / E2 and F1 / F2 contain data for 1 and 2 copies of non-internalizing, ADRBI Arg389Ser49 and Gly389Ser49 haplotypes, respectively. Event rates in the Placebo and Bucindolol Arms, HzR (C.I.s) and P values are given for comparison to the "All Subjects", all-genotypes data in Row A, and ADRBI Arg389Arg or Gly389 carrier genotypes in Rows B 1 or Cl, respectively. For both clinical endpoints differential efficacy for ADRBI Arg389Arg homozygous vs. Gly389 carrier genotypes is evident, with HzRs statistically significantly reduced in the former (0.60 ACM / Tx, 0.58 AF / AFL / ACM) but not in the latter (0.93, 0.80 respectively), consistent with enhancement of the bucindolol treatment effect in subjects with a ADRBI Arg389 homozygous genotype.23When 2 copies of Ser49 containing haplotypes are compared to 1 copy, HzRs for Arg389Ser49 and Gly389Ser49 are progressively reduced, for both clinical endpoints (FIG. 34, Rows El, 2 and Fl, 2). When ADRB2 "Not Gln27Argl6" noninternalizing haplotypes (>1 copy) are added to the 2 copy ADRBI non-internalizing groups in FIG. 34 (Rows E3 and F3) HzRs are further reduced for both Arg389Ser49 and Gly389Ser49 in the ACM / Tx endpoint, although there is no further reduction for AF / AFL / ACM. Arg389Gly49 haplotypes (Rows D2, D3) do not exhibit these patterns, for either endpoint. However, for both ACM / Tx and AF / AFL / ACM placebo event rates drop substantially proceeding from All Subjects to 1 copy and then 2 copies of Arg389Gly49 (FIG. 34, Rows A and D), 22.3 / 18.0 / 11.1% for ACM / Tx and 28.8 / 26.2 / 11.1% for AF / AFL / ACM, consistent with a gene-dose effect of the Gly49 internalization variant.ADRBI and ADRB2 Haplotype Effects Are Equivalent in Black and Non-Black Race

[0172] As shown in Tables 1 and FIG. 7, African-ancestry confers a greater degree of common genetic variation within fl-ARs, as in other neurohumoral31and non-neurohumoral systems.32FIG. 5 consists of bucindolol vs. placebo time to event curves in the All LVEFcohort by Black or non-Black racial categories for the composite endpoints of time to first CVM / HFH and CVH, in subjects with Double Internalizing (>2DI, and Not Double Internalizing (>2NI all= other ADRB1 and ADRB2) haplotypes. For the >2DI haplotype condition there is no evidence of a bucindolol treatment effect in either Blacks or non-Blacks, with HzRs ranging from 0.84 (CVM / HFH in non-Blacks) to 1.75 (CVM / HFH in Blacks). In contrast, in >2NI subjects for both endpoints there is evidence of a treatment effect, with little or no difference in HzRs between Black and non-Black subjects (HzRs ranging from 0.64 (0.39, 1.06) for CVH in Blacks to 0.76 (0.61, 0.96) for CVH in non-Blacks). These data indicate that within the >2NI haplotype phenotype there are no differences between Blacks and nonBlacks for enhancement of bucindolol treatment effects.

[0173] The haplotype dose response progressing from >2 Double Internalizing to >2 Noninternalizing in placebo treated patients (Figures 1, 7) is also observed in Blacks (FIG. 35). For both non-Blacks and Blacks, the 7 endpoint ORs in (Tier 1) >3DI subjects are consistently to the left of the line of identity, Tier 3, >3DNI point estimates are to the right of 1.0. and 21 Incompletely Internalizing estimates are intermediate and around 1.0.Internalizing / Non-internalizing Haplotypes in GENETIC-AF

[0174] The GENETIC-AF trial, a Phase 2 trial that exclusively randomized HFrEF and HFmrEF (HF with LVEF 0.40-0.49) patients who were ADRB1 Arg389Arg genotype (N=267) and at risk for developing recurrent AF,26included a voluntary DNA Bank substudy that enrolled 138 randomized patents who were subsequently genotyped for ADRB1 Ser49Gly and ADRB2 position 27 and 16 polymorphisms. Baseline characteristics by diplotype for this cohort are in FIG. 15. The small sample size of the DNA substudy precluded assessment of haplotype impact on clinical events, but the trial included peripheral venous plasma NT -proBNP and NE biomarker measurements at baseline (pre-randomization to bucindolol or metoprolol succinate) and at 4, 12 and 24 weeks (trial end) of efficacy follow-up.26The genotypes, haplotypes and diplotypes are in FIG. 16.

[0175] In order to investigate possible dose-response of [3-AR haplotype interactions with treatment for effects on NT-proBNP and NE in this cohort, subject diplotypes were divided into >2DI and >2NI groups as for the BEST substudy, and biomarker changes relative to baseline were assessed at 4,12 and 24 (FIG. 6, 17-20). Biomarker results were also measured in 4DNI diplotype subjects (FIG. 17-20). Baseline characteristics within ADRB1 and ADRB2 diplotype cohorts (FIG. 31) or in the treatment arms do not differ meaningfully. NT-proBNPis a biomarker for both AF and HF,26and thus is a suitable surrogate for GENETIC-AF's primary endpoint of time to symptomatic AF / AFL / ACM in a HF population. In bucindolol- treated subjects with 4DNI haplotypes, compared to a baseline value of 1304+1313 (mean+sd) pg / ml (FIG. 31), at 4, 12 and 24 weeks NT -proBNP is decreased by respective amounts of 737+312 pg / ml (mean+sem, P= 0.009), 493+329 pg / ml (P=0.15) and 778+266 pg / ml (P= 0.005). Metoprolol treated patients with 4DNI haplotypes have a baseline NT-pro-BNP value of 1171+292 pg / ml (FIG. 18), and in contrast to the bucindolol group exhibit no statistically significant changes at 4, 12 and 24 weeks (FIG. 6, FIG. 18). For ADRB1 and ADRB2 >2DI haplotypes, from respective baseline values of 1068+1049 pg / ml (mean+sd) and 670+308 pg / ml in the bucindolol and metoprolol groups (FIG. 17-18), neither exhibit changes in NT- proBNP at any timepoint (FIG. 6, 17, 18). In >2NI haplotypes, in the Bucindolol Group NT- proBNP is reduced from a baseline of 1364+1494 (mean+sd) (FIG. 17) by 445+147 pg / ml (mean+sem, P<0.001), 361+158 pg / ml (P=0.043) ,and 380+143 pg / ml (P=0.003)at 4 , 12 and 24 weeks respectively(FIG. 6, 17). Metoprolol-treated patients with >2NI haplotypes have no significant changes from baseline (FIG. 6, 18).

[0176] NE measurements are shown in FIG. 6 and in FIG. 19 and FIG. 20. For bucindolol- treated subjects with 4DNI haplotypes the pattern is similar to NT-proBNP; from a baseline of 631 pg / ml (mean+sd) there are statistically significant (P values 0.01 to 0.026) reductions by 147+50.6 (mean+sem), 158+55.8 and 140+43.3 pg / ml at 4, 12 and 24 weeks, respectively (FIG. 6, FIG. 19). Also similar to NT-proBNP, in 4DNI haplotypes there are no reductions in NE at any timepoint in the Metoprolol Arm (FIG. 6, FIG. 20). Results for >2DI Double Internalizing haplotypes are also similar to NT-proBNP, with no significant changes at any timepoint in either the Bucindolol or Metoprolol Arms (FIG. 6, 19 and 20, respectively). The >2NI, Bucindolol Group (FIG. 6, 19) results are also similar to those for NT-proBNP, with statistically significant reductions from baseline (738+362 pg / ml (mean+sd) at 4 weeks (by 174 pg / ml (mean+sem, P<0.001, at 12 weeks (by 182 pg / ml, P<0.001) and by 136 pg / ml at 24 weeks (P=0.001). In the metoprolol arm >2NI subjects there are no changes in NE at any timepoint. (FIG. 6, 20).Expansion of the Bucindolol Pharmacogenetic Target Population and Efficacy by the Inclusion of ADRB1 , ADRB2 Non-internalizing Haplotypes

[0177] The clinical outcomes efficacy enhancing effects of non-internalizing haplotypes on bucindolol vs. placebo treatment effects in the BEST substudy create the potential forenlarging bucindolol’s pharmacogenetically targeted population beyond ADRB1 Arg389Arg. Also, within the expanded target population there is the possibility that newly identified pharmacogenetic subgroups might yield treatment effects superior to current ADRB1 Arg389Arg target enhancement.23,26FIG. 21, 22, 36 contain these analyses, based on data from the LVEF >0.20 and All-LVEF cohorts. For the LVEF >0.20 cohort treatment effects for all listed pharmacogenetic groups (Columns 2-8 in FIG. 21) exceed that for the All-Genotypes parent cohort (Column 1), at a significance level of at least P <0.05. In addition, in 4 of the 6 non-internalizing diplotype-containing subgroups (Columns 3-8) treatment effects exceed those of the Column 2 ADRB1 Arg389Arg genotype index pharmacogenetic enhancement subgroup. Moreover, the combined ADRB 1 AND ADRB2 Maximum Double Non-internalizing (4DNI) diplotype subgroup (Column 6) treatment effect exceeds the Column 2 index group by 1.34 fold (P=0.0017). The combination of the 4DNI subgroup with non-overlapping ADRB1 Arg389Arg genotype subjects (Column 7) creates a subgroup that is 33% larger than the Column 2 index pharmacogenetic group (N=441 vs. 331), with a slightly higher (by 1.03 fold, P=O.O38) treatment effect on the paired endpoints analysis. Finally, the requirement that both the ADRB1 Arg389Ser49 homozygous diplotype AND non-intemalizing ADRB2 diplotypes be present (Column 8, 10% of the total cohort) creates an increase in treatment effect of 1.52 fold >Column 2, and 1.89 fold >Columnl.

[0178] Analogous data for the All-LVEFs cohort are in FIG. 22, 36. The results are similar to those in FIG.21, and yield respective Column 8 (11% of the cohort) treatment effect enhancements of 2.06 fold vs. Column 1 (P <0.01), and 1.77 fold vs. Column 2 (P<0.01).ERK1,2 Phosphorylation in Human Isolated RV Trabeculae

[0179] In order to confirm that bucindolol acts as a biased ligand in the human heart and is capable of activating a putative cardioprotective pathway via P-AR receptor internalization, the inventors assessed ERK1,2 phosphorylation in preparations of RV trabeculae isolated from 4 explanted failing hearts that had at least 1 copy of ADRB1 and ADRB2 non-intemalizing haplotypes. After a 5 or 10 minute tissue bath incubation, bucindolol is associated with increased ERK1,2 phosphorylation compared to vehicle or metoprolol, and at 60 min dephosphorylation occurs (FIG. 37). Similar results were obtained in 3 other preparations.DISCUSSION

[0180] For same-allele nonsynonymous SNPs the encoded product that includes each polymorphic locus has the potential to modify function, as it is the protein haplotype thatconfers the net biologic effect.28-30,33For example, the fh-AR position 389 amino acid polymorphism contains an Arg or a loss of function Gly that dictates a major difference in signal transduction capacity and agonist affinity,22,23,34but receptor function is also influenced by a position 49 polymorphism that regulates ligand-mediated internalization. This means that the ADRB1 389 / 49 haplotype can encode a higher function Pi-AR that easily internalizes (Arg389Gly49),21a higher function receptor relatively resistant to internalization (Arg389Ser49), and a lower function receptor resistant to internalization (Gly389Ser49). The 4th possibility, Gly389Gly49, would theoretically have lower function and would easily internalize, but was not found in this and most other genotyping studies,21,35presumably due to strong linkage disequilibrium. The p - AR does not have a polymorphic locus that modulates signal transduction capacity or agonist binding affinity, but has 2 loci that act in tandem to regulate internalization, with Gln27Argl6 exhibiting a high level of internalization19or rapid desensitization20to the agonist isoproterenol. Similar loADRBl, only 3 ADRB2 haplotypes are usually found in genetic studies including in the current report, with Glu27Argl6 absent. The missingness of 1 of the 4 possible haplotypes in each fl-AR gene allows haplotypes to be determined with near certainty from genotype data. In the study, for both AD RBI and ADRB2 double heterozygote genotypes that would theoretically give rise to 2 double heterozygote diplotypes, the existence of only 1 for each gene (respectively Arg389Gly49 / Gly389Ser49 and Gln27Argl6 / Glu27Glyl6) due to the respective absences of the Gly389Gly49 and Glu27Argl6 haplotypes was confirmed by long-read DNA sequencing.ADRB1,ADRB2 Haplotypes in the BEST Adrenergic Receptor Polymorphism Substudy.

[0181] The data from the BEST Adrenergic Receptor Polymorphism pharmacogenomic substudy in both the greater (All LVEF) and lesser (LVEF >0.20) EV dysfunction cohorts indicate that placebo treatment of advanced HFrEF results in reduced HF and AF outcome event rates in patients who have ADRB1 and ADRB2 haplotypes that encode readily internalizing receptor variants. In a HF patient such receptor internalization could occur on exposure to increased levels of NE, which was present in both investigated clinical trial patient populations. In the BEST substudy, across 7 clinical endpoints placebo-treated subjects with intemalizing(>3DI) haplotypes had lower clinical event rates and ORs <1.0 compared to counterpart haplotypes, whereas patients with non-internalizing (>3DNI) haplotypes exhibited higher event rates and ORs >1.0 vs. counterparts. In contrast, subjects in the Bucindolol Group exhibited more uniform effects across internalization tiers, with no statistically significant ORsbetween internalization tier haplotypes and their counterparts. As a result of these within- treatment group between-haplotype tier effects, on unadjusted analyses bucindolol vs. placebo event rate ORs were lower in subjects with >3DNI haplotypes, trended lower in Incompletely Internalizing (21) subjects, and trended higher in patients with >3DI haplotypes. Subject level structural equation model covariate adjustment confirmed and extended the unadjusted analyses, as on progression from internalizing to non-internalizing haplotypes the majority of 7 clinical endpoints in placebo treated subjects exhibited an increase in in event rates, bucindolol treatment was associated with no changes across haplotype tiers, and the bicindolol vs. placebo treatment effects exhibited a rate reduction in 13 of 14 endpoints in the 2 investigated cohorts.

[0182] The between internalizing haplotype groups and event rate OR data were further corroborated by time to clinical event analyses, where placebo treatment resulted in a reduction in HzR in subjects with internalizing (>2DI) compared to non-internalizing (>NI) haplotypes. In contrast, for bucindolol treatment there was no difference (HzRs approximately 1.0) in subjects with >2DI vs. >NI haplotypes, with time to event rates similar to placebo treatment in the >2DI haplotype group. That is, when there is at least 1 internalizing haplotype for ADRB1 and ADRB2, placebo (or presumably no treatment) has a favorable effect equivalent to bucindolol, which is only effective vs. placebo in subjects with >2 non-internalizing ADRB1 a ov ADRB2 haplotypes. The preference of bucindolol for non-internalizing [3- AR haplotypes was underscored by results in subjects with these variants exclusively (4DNI), who had vs. placebo clinical event HzRs from 0.17 to 0.49 equal to event rate reductions from 51% to 83%.

[0183] Therefore, compared to placebo the biased ligand P-blocker bucindolol conferred a more favorable treatment effect in subjects with non-internalizing haplotypes, which constituted approximately 70% of the BEST substudy entire cohort. These data suggest that, in the absence of biased ligand signaling, on increased P-agonist exposure as occurs in the failing heart, easily internalizing fJ-ARs mediate beneficial effects on HF and AF clinical events, similar to what has been reported for prevention of ventricular fibrillation in acute MI.21In contrast, non-internalizing [3-ARs confer a harmful effect that can be abrogated by a [3- antagonist, with potential treatment effect enhancement by a biased ligand [3-blockcr.ADRB1 , ADRB2 Haplotypes in the GENETIC-AF Trial

[0184] The limited sample size of the GENETIC-AF DNA substudy was nevertheless sufficient to detect fJ-AR haplotype effects on NT-proBNP and NE. In the parent GENETIC- AF cohort, compared to metoprolol both NT-proBNP and NE systemic plasma levels were reduced by bucindolol,26,36and the in the current study the reductions in NT-proBNP or NE in the bucindolol group occurred only in subjects with 4DNI or >2NI non-internalizing haplotypes. In contrast, metoprolol succinate was not associated with a reduction in NT- proBNP or NE in subjects with non-internalizing or internalizing haplotypes.Haplotype Data Indicate that Both ADRB1 and ADRB2 Internalizing Variants Affect Heart Failure Clinical Events and Bucindolol Treatment Effects.

[0185] Human cardiac myocytes contain f -ARs that are coupled to positive inotropic effects via the canonical camp / PKA pathway,1and to ERK1,2 signaling following internalization.10When overexpressed in mouse hearts human f -ARs mediate histopathological effects,2,37and in failing human ventricles the proportion of fn-ARs / Total [3- ARs is increased, from 20-25% to 35-40%.1However, the numerically dominant fh-AR signaling pathway is more biologically adverse, and in clinical trials mortality reduction outcomes are similar for f -AR selective blocking agents and the nonselective, biased ligand P-blocker carvedilol.1Despite these observations, in the current study non-internalizing f -AR receptor haplotypes were associated with improved clinical outcomes with bucindolol treatment, quite similar to the enhancement conferred by non-internalizing ADRB1 haplotypes. This suggests that myocardial f -ARs are operational in the natural history of adrenergically- mediated pathologic ventricular remodeling, and that their pharmacologic manipulation can have therapeutic consequences. Moreover, for clinical endpoints the combination of ADRB1 and ADRB2 non-internalizing diplotypes was additive, as might be expected based on the known differences in their receptor signaling pathways.1By-Race Data

[0186] As for the ADRB1 Arg389Gly polymorphism,31race also affects [3- AR internalizing variants, with Blacks compared to non-Blacks generally having higher frequencies for internalizing haplotypes and lower frequencies of non-internalizing haplotypes. This means that effects of placebo or nonbiased [3- AR ligands favor Blacks, extrapolating from the genedose effect of internalizing ADRB1 and ADRB2 haplotypes on clinical and biomarker outcomes. In contrast, some of bucindolol’ s efficacy enhancement is dependent on the presenceof non-internalizing haplotype encoded receptors, for which in the BEST pharmacogenomic substudy non-Blacks had a slight advantage in ADRB1 Ser49 and ADRB2 non-Gln27Argl6 containing haplotypes. However, as shown in the current and a previous genotype study,31within the same efficacy enhancing haplotype or genotype Blacks have therapeutic responses to bucindolol that are equivalent to those in non-Blacks.Biased Ligand Properties of Bucindolol

[0187] ERK1,2 phosphorylation experiments in isolated human heart preparations suggest that the favorable effects of bucindolol in relatively internalization resistant receptors may have been due to "cardioprotective" signaling6,21mediated by biased ligand-induced receptor internalization. A biased ligand can be defined as a compound that on receptor binding can signal through both G-protein dependent and independent pathways.37In the context of the current study, cell surface Pi- or i-ARs act through the G-protein-cAMP pathway, and internalized receptors through MAP kinase / ERKl,2. As it applies to bucindolol or the structurally similar compound carvedilol,38in systems that are hypersenstive for the detection of G protein mediated adenylate cyclase stimulation / cAMP generation both compounds exhibit weak canonical -agonist activity, but also can active MAP kinase ERK1,2 signaling independent of G-proteins.14For both biased ligands the P-agonist activity is insufficient for activation of Pi- or P2-AR pathways in the human heart, either in vitro23,39,40or in vivo.41,42Prior data supporting bucindolol's biased ligand signaling in Pi- and Pi-ARs includes, in comparison to carvedilol,6,13,15,43similar ligand binding characteristics that differ from other P- AR antagonists when co-crystallized with the avian Pi-AR,38higher potency of bucindolol for ERK1 / 2 activation,14similar agonist / guanine nucleotide modulatable binding (a characteristic of agonists) in human Pi- and P2- ARs,39,40similar ability to activate adenylate cyclase in certain model systems,14and for bucindolol in contrast to carvedilol downregulation of Pi- and P2-ARS after 24 hours of isoproterenol incubation with no reduction in cognate mRNA (evidence of internalization leading to receptor degradation from lysosomal sorting44,45). These latter experiments were performed in both cultured avian cardiac myocytes (primarily Pi-ARs and hamster ADRB2 smooth muscle cells (P2-ARS) that have non-internalizing P-AR genotypes.46-48In addition, there is one report of bucindolol and carvedilol having similar inhibition of ERK1 / 2 activation by endocytosis / internalization inhibitors.49In the current study the bucindolol data for ERK1,2 phosphorylation in isolated human heart preparations with noninternalizing ADRB1 and ADRB2 diplotypes support these previous data, indicating thatbucindolol has biased ligand activity capable of increasing internalization of a noninternalizing Pi- or 2-AR haplotypes. In comparison, based on biomarker and ERK1,2 activation data the nonbiased ligand -blocker metoprolol did not exhibit such haplotype specificity.

[0188] Carvedilol’ s interaction with - AR haplotypes has not been previously investigated, but despite their similarities bucindolol and carvedilol also have somewhat different properties including sympatholysis with bucindolol1,34,50but not with carvedilol1,50that may account for the former's effectiveness enhancement in HF patients who are homozygous for ADRB1 Arg389.1,23Unlike bucindolol, the biased ligand P-blocker / vasodilator carvedilol has not exhibited selective enhancement of transplant free survival in subjects with ADRB1 Arg389Arg51,52or Ser4951containing genotypes. Bucindolol is also a more potent P2-AR antagonist than carvedilol, by approximately 5-8 fold.40In addition, differences in biased ligand potency in favor of bucindolol14could be a factor for differential efficacy enhancement in noninternalizing Pi- or P2-ARS. Therefore, the absence of any P-AR internalization pharmacogenetic clinical data with carvedilol precludes speculation on its effects on noninternalizing vs. internalizing P-AR haplotypes.Haplotype Targeting Expands the Pharmacogenetic Heart Failure or Atrial Fibrillation Prevention Potential of Bucindolol

[0189] In BEST substudy subjects with LVEFs >0.20 the enhanced treatment effects of bucindolol in subjects with both ADRB1 AND ADRB2 Maximum Double Non-internalizing (4DNI) diplotypes resulted in a 1.34 fold increase in treatment effects compared to the ADRB1 Arg389Arg index population, and 1.69 fold compared to the All-Genotypes cohort. If ADRB1 Arg389Arg genotype AND 4DNI diplotypes were considered (a 10% subgroup) the treatment effect enhancement compared to the index ADRB1 Arg389Arg group was by 1.52 fold in the LVEF >20% cohort and by 1.40 fold in the All LVEFs cohort, with respective increases vs. the All-Genotypes cohort of 1.91 and 2.03 fold.

[0190] When the ADRB1 AND ADRB2 >4DNI subgroup was combined with ADRB1 Arg389Arg subjects by OR logic the result was a larger subgroup (by 32%-33%) that was essentially identical to ADRB1 Arg389Arg for treatment effects, in both LVEF cohorts. As a result, in the BEST pharmacogenomics substudy the addition of non-intemalizing ADRB1 and ADRB2 haplotypes (“haplotype targeting”) to the current ADRB 1 Arg389Arg targeting resultedin a larger population eligible for benefit, increasing from 45-47% to 60-62% of the allgenotype parent population. Since the BEST pharmacogenetic substudy was enriched in African- Ancestry (22% compared to 13% in the U.S. population) and Blacks have a lower frequency of both ADRB1 Arg389 and non-internalizing AD RBI and ADRB2 haplotypes, eligibility for therapeutic enhancement in the entire U.S. population would increase from 49% to 65%. Thus by using the ADRB1 Arg389Arg OR (ADR J AND ADRB2 Non-internalizing) algorithm the population eligible for treatment enhancement can be increased by 33%.Natural History Implications

[0191] The effects in the placebo-treated group could have implications for heart failure natural history, which in the absence of other prognostic factors would be expected to be more favorable in Blacks due to their higher frequency of the internalizing haplotype variants of ADRB1 Arg389Gly49 (by 1.71 fold) and ADRB2 Gln27Argl6 (by 1.21 fold). Although one recent study of HF prognosis in Blacks vs. non-Blacks demonstrated a survival advantage in Blacks,53other studies54have shown worse outcomes of Blacks with HF. In any case, the effects of P-AR internalizing variants on HF prognosis deserves further epidemiologic investigation, with a focus on racial differences in the fl-AR polymorphisms that affect HF natural history.Conventions and Presentation Formats

[0192] Two loci haplotypes are presented in an allelic orientation as (encoded amino acidposition 1- amino acid-position2, e.g. ADRB1 Arg389Gly49. Diplotypes (aggregate haplotypes from each allele) are written as allele 1 (amino acid-position / / allele 2 (amino acid-position 1- amino acid-position 2), e.g. for ADRB1 Arg389Gly49 / Gly389Ser49, or alternatively as in tables to emphasize their allelic non-alignment. Two loci genotypes are presented with the Arg389Gly49 / Gly389Ser69 encoded amino acids flanking their position number and separated by a comma, e.g. Arg389Arg, Ser49Gly. Diplotypes are described as homozygous (two identical alleles in each haplotype), double heterozygous (each of the 2 positions comprised of different encoded amino acids), or single homozygous (1 position with the same encoded amino acid on both alleles, the other position with different amino acids). Polymorphisms are presented as (encoded amino acid-position-counterpart amino acid), and annotated as a "polymorphism" to differentiate from a heterozygote genotype designation, e.g. "Arg389Gly polymorphism" vs. "Arg389Gly genotype". ADRB1 and ADRB2 haplotypes are designated as"internalizing" and "non-internalizing" to categorize them as having a respective greater or lesser degree of internalization on exposure to agonists.

[0193] Clinical event-haplotype dose-response analyses were assessed by comparing subjects with internalizing vs. non-internalizing ADRB1 and ADRB2 variants. Because of smaller sample sizes (internalizing haplotypes, Black race) or genotype restrictions GENETIC- AF cohort) the definition of internalizing groups varied by the specific analysis, but always consisted of more vs. fewer internalizing haplotypes. As an aid to comparing these analyses, each defined comparator was assigned a unique descriptor. Using Odds Ratios as the readout, from the BEST adrenergic receptor polymorphisms N=1040 pharmacogenomic substudy ADRB1 OR ADRB2 haplotype combinations are categorized as: "Double Internalizing" (>3DI, 3-4 internalizing haplotypes, Tier 1); "Incompletely internalizing" (211, 2 internalizing haplotypes, Tier 2), or "Double Non-internalizing" (>3DNI, 0-1 internalizing haplotypes, Tier 3). For time to event (Kaplan-Meir) curves generating hazard ratios (HzRs) for clinical events in the BEST substudy haplotype groups were subdivided into “Double Internalizing (DI)” (>1 copy of ADRB1 AND ADRB2 internalizing haplotypes), “Double Non-internalizing (NDI)” (all other ADRB 1, ADRB2 haplotype combinations), or “Maximum Double Non-Internalizing” (MDNI) (2 copies of ADRB1 AND ADRB2 non-internalizing haplotypes, no copies of internalizing haplotypes. For the GENETIC-AF N=138 pharmacogenetic biomarker substudy Double Internalizing (DI) was defined as for BEST HzR analyses (>1 copy of ADRB1 AND ADRB2 internalizing haplotypes), but the BEST NDI group was subdivided into Incompletely Internalizing (III), (1 copy of ADRB1 OR ADRB2 internalizing haplotype) and Maximum Double Non-internalizing (MDNI), 2 copies of ADRB1 AND ADRB2 internalizing haplotypes, no copies of internalizing haplotypes, the same definition as for the BEST HzR analysis).Summary

[0194] The presented evidence for the biased ligand P-blocker bucindolol enhancing treatment effects via internalization of relatively non-internalizing fl-ARs consists of 1) [3- AR haplotype-referenced extensive clinical endpoint and biomarker data from 2 clinical trial pharmacogenetic substudies, 2) the linkage of these data to previously reported [3- AR haplotype internalization or internalization proxy data, and 3) the demonstration in isolated human heart preparations that a consequence of biased ligand promoted [3- AR internalization, ERK1,2 activation,8 10 12,55occurs in cardiac tissue with non-internalizing ADRB1 and ADRB2 haplotypes. However, in addition to the biased ligand / receptor intemalization / cardioprotectivesignaling hypothesis there are other possible explanations for bucindolol’s enhanced treatment effects on non-internalizing fl-ARs. These include non-MAPK / ERKl,2 differential intracellular signal trafficking of encoded haplotypes,56and internalization-based exclusion of cell surface fl-ARs from adverse canonical signaling.Conclusions

[0195] In HFrEF and HFmrEF patients polymorphic loci in ADRB1 and ADRB2 that encode protein haplotypes regulating receptor internalization affect HF and AF clinical events as well as biomarkers, in both placebo- and bucindolol-treated subjects. Haplotype pharmacogenetic targeting is feasible in HF, and can identify populations at differential risk for serious outcomes as well as candidates for increased therapeutic effectiveness.STAR METHODSCLINICAL TRIAL OR EXPLANTED HEART STUDY POPULATIONS

[0196] The study cohorts consistent of patients with heart failure (HF), from the “Pharmacogenomics of ^-Adrenergic Receptor Polymorphisms and Response to P-blockers in Heart Failure” substudy (“BEST Pharmacogenomics Substudy”23of the Beta-Blocker Evaluation of survival Trial (BEST),27and the GENETIC-AF (Genotype-Directed Comparative Effectiveness Trial of Bucindolol and Toprol-XL for the Prevention of Symptomatic Atrial Fibrillation / Atrial Flutter in Patients with Heart Failure) trial.26The primary endpoints of these studies were respectively: time to all-cause mortality or cardiac transplantation (ACM / Tx);23and time to first atrial fibrillation or flutter, or ACM (AF / AFL / ACM).26In both studies DNA was extracted from peripheral whole blood using standard techniques.BEST Trial (NCT00000560) Pharmacogenomic Cohort

[0197] The 1040 patient Adrenergic Receptor Polymorphism substudy23and its parent trial27have been previously described, including the BEST trial protocol details and a consort diagram.58The primary endpoint of the parent trial was time to all-cause mortality (ACM), with secondary endpoints of time to: 1) ACM or cardiac transplantation (ACM / Tx), cardiovascular hospitalization (CVH), and heart failure hospitalization (HFH). At FDA request after completion of the trial time to cardiovascular hospitalization (CVH) was also measured,34and the composite of time to CVM / HFH, now the preferred primary endpoint in Phase 3 trials, has also been analyzed and reported.59BEST randomized an advanced HFrEF (Heart Failurewith reduced LV Ejection Fraction) population (NYHA Class III and IV, with LVEFs <0.35). The trial was sponsored by the NHEBI, and with the exception of 2 Canadian centers was conducted in the U.S, with the study population purposely enriched for Sub-Saharan African- Ancestry (22% in the substudy23). The race / ethnicity case report form classifications were “White, Black, Hispanic, Asian / Pacific Islander, American Indian / Alaskan, Other” determined by self-identification. In the current as well as previous reports,27Blacks were assigned one racial category, and all other races were classified as non-Blacks. Sex reporting was by selfidentification as recorded on case report forms. BEST enrolled an advanced heart failure in the adrenergic receptor polymorphisms substudy respectively 93 % and 7% NYHA Class III and IV, FIG. 7) and moderate-severe EV dysfunction mean (EVEFs 0.24+0.07), and in addition to this 1040 patient all-LVEF parent cohort an N=723 subgroup with less severe LV dysfunction (>0.20, mean 0.27+0.05; Class III / IV 93% / 7%%) was also analyzed. The BEST parent trial was approved by ethical committees at the NHLBI, VA Cooperative Studies Program, the BEST Trial DNA Oversight Committee, and each investigative site. All patients signed written informed consent for both the parent trial and the DNA substudy. Adverse events for the parent trial have been previously reported, and there were no adverse events related to DNA sample collection.

[0198] Sample size in the BEST pharmacogenetic substudy was originally for 1000 randomized patients required to achieve 90% power for detection of a 20% effect size with a P <0.05 between bucindolol and placebo for the primary endpoint of ACM / Tx, for any of the 5 polymorphic loci in the original application (ADRB1 Arg389Gly, Ser49Gly; ADRB2 Gln27Glu, Glyl6Arg; ADRA2C 322-325 Indel). To account for multiplicity of the 5 loci the critical value was set at 0.010, and for ADRB1IADRB2 internalizing vs. non-internalizing haplotypes a Bonferroni correction would be P=0.025.GENETIC-AF (NCT01970501) Cohort

[0199] GENETIC-AF was a 267 subject Phase 2 trial in HFrEF and HFmrEF (HF with mildly reduced LV Ejection Fraction, defined as LVEFs between 0.40 and 0.49) patients with a history of persistent or paroxysmal AF, randomized to bucindolol or metoprolol succinate for AF prevention after spontaneous or electrical cardioversion attainment of sinus rhythm.26The protocol details60including a consort diagram26have been previously published. The primary endpoint of GENETIC-AF was time to atrial fibrillation, or atrial flutter, or ACM (AF / AFL / ACM), and norepinephrine (NE) and NT -proBNP were tertiary endpoints.26Thismultinational trial included pre -randomization genotyping for ADRB1 Arg389Gly and only randomized Arg389Arg subjects;26138 of which subsequently had ADRB1 Ser49Gly and ADRB2 genotypes determined via an elective DNA substudy.60Data from the race / ethnicity case report form was also converted into Black and non-Black classifications, and sex reporting was by self-identification reported on case report forms. Baseline characteristics by diplotype combinations are in FIG. 20. The sample size estimate for the trial was N= and 330 primary events for the primary endpoint of time to first symptomatic atrial fibrillation / atrial flutter or all-cause mortality episode (AF / AFL / ACM), but based on its “seamless” Phase 2-3 design the trial was stopped in Phase 2B at 267 patients randomized with 143 primary events when a scheduled interim analysis indicated that the Bayesian predictive probability of success was below 10%.26As tertiary endpoints NT-ProBNP and NE plasma levels were not subjected to sample size estimates, but the actual sample size is well above the N required for demonstrating statistical significance in NE levels.36,49Adverse events for the parent trial have been previously reported,27and there were no adverse events related to DNA sample collection.Isolated Human Heart Studies

[0200] Explained failing hearts from were obtained at the time of cardiac transplantation from end stage heart failure patients with nonischemic cardiomyopathies, whose relatives or legal representative had given written informed consent for donation of tissue for research purposes.METHODS DETAILSGenotyping and Haplotyping.

[0201] BEST DNA substudy genotyping for ADRB1 Arg389Gly, ADRB1 Ser49Gly, ADRB2 Gln27Glu, and ADRB2 Glyl6Arg was conducted by Restriction Fragment Length Polymorphism (RFLP) conducted in DNA extracted from whole blood as previously described,23,61with results subsequently confirmed by DNA sequencing. For GENETIC-AF subjects the ADRB1 Arg389Arg genotype necessary for trial enrollment was determined by TaqMan® SNP genotyping as previously described,26while the ADRB1 Ser49Gly genotype and ADRB2 genotypes were measured by sequencing SNP flanking-primer PCR amplicons using DNA extracted from the ADRB1 Arg389Gly genotyping. Haplotyping in both substudies was by genotype imputation and likelihood methodology, using the “Haplo. stats” package available in R.21,62Linkage disequilibrium was tested using the R based "genetics package" Haplotypes for pairwise SNPs are onlyambiguous in the double heterozygote case; thus, for both ADRB1 and ADRB2 20 double genotype heterozygote DNA samples were subjected to long-read sequencing63that confirmed the likelihood reads of missingness of 1 haplotype from each gene (ADRB1 Gly389Gly49 and ADRB2 Glu27Argl6).Isolated Human Heart Studies

[0202] Failing hearts from end stage heart failure patients with nonischemic cardiomyopathies were obtained at the time of cardiac transplantation were immediately transported to the laboratory in iced Tyrodes’ solution. Right ventricular trabeculae (N=10-14) of uniform size (1-2 x 6-8 mm) were mounted in tissue baths and field stimulated to contract at 60 bpm at a bath temperature of 37° C, as previously described.23Following a 1 hour incubation period 1-2 trabeculae / group were incubated with vehicle, isoproterenol (lumol / L), bucindolol HCL(lumol / L) or metoprolol tartrate (10 umol / L) for either 5, 10, or 60 minutes. For the 60 minute incubations bath solution was changed every 15 minutes, with reconstitution of the incubating drugs. At the end of its incubation period each trabeculum was removed from the bath, flash frozen in liquid nitrogen and stored at -80°C. pERKl,2 Immunoblotting

[0203] Frozen trabeculae (25-35 mg) were homogenized in 25 volumes of a homogenization buffer cocktail (8M Urea, 2.5M thio-Urea, 4% CHAPS, 2mM EDTA, 0.01M dithiothreitol, 1% Protease Inhibitor Cocktail (Sigma P-8340) and 1% ReadyPrep TBP Reducing Agent (BioRad 1632101)) in glass mortar and pestle tissue grinders on ice. Supernatant fractions were saved after a 13,600xg centrifugation, aliquoted and frozen at - 80°C. Total protein content of the supernatants was determined by a Bradford assay (Pierce Detergent Compatible 1863028).

[0204] Immunoblots were performed on 5 micrograms of protein per sample by SDS- PAGE separation in 10% polyacrylamide gels (BioRad, Mini PROTEAN TGX 4561036), then transferred to 0.2 pm PVDF membranes (BioRad, ImmunBlot 16201777). Primary antibodies for ERK (Cell Signaling 9102), pERK (Cell Signaling 9101) and GAPDH (Santa Cruz 32233) were applied sequentially to the blots with the subsequent appropriate HRP-linked secondary antibody following (Sigma A2304 and A9169). Chemiluminescence was initiated (ThermoScientific SuperSignal West Pico-plus 34580) and captured by exposure to film that was then scanned and imaged by software package (ImageJ, NIH, imageJ.org). Chemical stripping of antibodies from the blot was performed between each primary antibody detection,before application of the next set of antibodies. pERKl,2 density was normalized to its respective unphosphorylated band, and compared to vehicle control at each incubation timepoint.QUANTIFICATION AND STATISTICAL ANALYSISAnalysis Strategy

[0205] The overall aim of the statistical analysis was to compare clinical event and biomarker outcomes in HF subjects with more vs. fewer internalizing fl-adrenergic receptor (f>- AR) haplotypes, across 2 clinical trials where haplotyping was performed and which had extensive outcomes databases. One of them, the BEST Pharmacogenomics Substudy,23was prospectively designed to detect potential differences by Black vs, non-Black race,27and the other was focused on recruiting a genetic population, ADRB1 Arg389 homozygotes,26who had previously been shown to be more responsive to the fl-blocker bucindolol.26We thus designed a statistical approach to be consistent with each trial’s original statistical analysis plan, but would also address the studies primary hypothesis that fl- AR haplotypes with different internalization potential affect HF outcomes. Because of variations in cohort sample sizes internalization subdivision definitions varied slightly between analyses (described in detail in the second paragraph of Supplemental Material, Conventions and Presentation Formats) but always included a comparison that encompassed >2 vs. <2 intemalizing / >3 noninternalizing (ADRB1 + ADRB2) haplotypes.BEST Adrenergic Receptor Polymorphisms Pharmacogenomics Substudy

[0206] The original statistical analysis plan (SAP) for the BEST Pharmacogenomics Substudy encompassed the measurement of 5 presumably independent polymorphisms: ADRB1 Arg389Gly, ADRB1 Ser49Gly, ADRB2 Gln27Gly, ADRB2 Glyl6Arg. The Thrl64Ile polymorphism was part of the original SAP, but was not analyzed due to its extremely low prevalence (minor allele frequency 0.12 (FIG. 8)). The ADRA2C 322-325 InDei was subsequently substituted,34leaving 5 polymorphisms for multiplicity consideration. In the original SAP statistical significance was defined as a P <0.05 for any of the 5 major vs. minor allele comparisons for the primary or secondary endpoints. To account for multiplicity in the 5 analyzed loci the critical value was set at P=0.010, or a standard Bonferroni adjustment. However, ss published thereafter21and shown in FIG. 10, the 4 fl-AR polymorphisms are not independent variables, since each of the 2 loci on ADRB1 and ADRB2 are in strong linkage disequilibrium.

[0207] The endpoints prospectively chosen for analysis in the current study included the primary cardiovascular endpoints for both the BEST Pharmacogenomic and GENETIC-AF DNA substudies (ACM / Tx and AF / AFL / ACM, respectively), the primary endpoint of the BEST parent trial (ACM) that was a secondary endpoint in its substudy, 2 secondary endpoints from the BEST parent trial (CVM, HFH),582 composite endpoints from these secondaries (CVM / HFH and ACM / HFH), and 1 post hoc endpoint (CVH) requested by the FDA. For graphical efficiency, in forest plots the ACM / HFH endpoint was not included. For the current study we performed 2 types of analyses on the combined ADRB 1 and ADRB2 haplotypes event rate and ORs rank ordered into 3 non-overlapping tiers by haplotype internalization potential, the first on the aggregated endpoints and the second a by-subject analysis with covariate adjustment. In the by-subject analysis non-cardiovascular mortality and non-cardiovascular hospitalization were also analyzed. These analyses were performed by treatment group, and on the treatment effects in the bucindolol vs. placebo group, using intention to treat data.

[0208] The aggregated endpoint analysis followed the general principles of the original SAP by designating an omnibus critical value of P <0.05 for all 7 endpoints on a Friedman (nonparametric) test, then applying a Dunn’s multiplicity test to determine which tier was different from another at a P <0.05 (FIG. 13). For subject level analyses a multi-group structural equation model (“SEM”) for binary outcomes was used to estimate the influence of internalization tier on cardiovascular and non-cardiovascular event rates by treatment arm. At the subject level the likelihood of occurrence of each of the seven candidate endpoints was modeled using Internalization Tier and the covariates EVEF, Coronary Artery Disease, Sex, Race, Volume Overload, NYHA Class, AF at Baseline, DNA Fag (time between randomization and blood draw for DNA extraction), Duration since HF Diagnosis, Age, Baseline SBP and Baseline HR (all covariates that have been used for stratification in the 2 clinical trials or have been shown to relate to the probability of occurrence of the primary endpoints23,26,27) (FIG. 14). This methodology allowed for estimation of the effect of internalization tier on each endpoint, simultaneously accounting for unique effects of each of the 12 covariates, as well as the degree of dependency between endpoints. An equivalently formulated model substituted the Internalization Tier predictor for Treatment, evaluating the by-endpoint estimated treatment effects within the > 3DNI group. The readouts and code for these analyses are archived in the GitHub site.

[0209] For the ADRB1 IADRB2 haplotype titration forest plots (Figures 26-31), constructed for pattern recognition and consistency between clinical endpoints, only the individual comparison OR 95% C.I.s and P values were calculated. For time to event (Kaplan-Meier) curves all analyses used unadjusted methodologies, as adjustments, particularly in small sample sizes, may yield misleading results. However, for sensitivity purposes we also performed analyses adjusted for randomization stratifiers and other covariates, which were not appreciably different from the unadjusted versions and are archived in the GitHub site.GENETIC-AF DNA Substudy

[0210] In the GENETIC-AF DNA substudy cohort the SAP26defined analysis for the tertiary endpoints of NT-proBNP or NE was a change from baseline values using nonparametric methods, since in previous studies these were known to be non-normally distributed. These analyses were performed on the trial’s efficacy cohort, defined as receiving blinded study medication or having it stopped within 2 weeks of biomarker collection.Statistical Test Details

[0211] Time to first event Hazard Ratios (HzR) were generated by unadjusted Cox proportional hazards models, with first clinical event Odds Ratios (ORs) obtained from logistic regression models. Bucindolol treatment effects based on event rates were calculated by (Bucindolol Event Odds) / (Placebo Event Odds). Confidence intervals for linkage disequilibrium coefficients were calculated using 10,000 nonparametric bootstrapped estimates. Treatment effects between bucindolol vs. control (placebo or metoprolol succinate) were calculated from HzRs as: % treatment effect = ((1-HzR) x 100). Data normal distribution was determined by the Anderson Darling test. For normally distributed data assessing one clinical endpoint t-tests or a one-way ANOVA with Holm-Sidak test was used. For non- normally distributed data Wilcoxon or a Friedman’s test with a Dunn’s multiplicity adjustment was employed. Growth curves of clinical endpoint aggregate event rates across internalization tiers were analyzed by beta regression.Example 2: Prevention of Atrial Fibrillation in Heart Failure Patients by Pharmacogenetic Targeting of Bl-Adrenergic Receptor Genetic Variants: Mechanisms, Target Precision and Clinical OutcomesPharmacogenetic Targeting

[0212] Pharmacogenetic targeting (PgT), a concept rooted in Ehrlich's "Magic Bullet" construct,23is a form of precision therapy where a genetic biomarker identifies patients in whom a favorable treatment response would be expected to exceed that of the general population.24,25The value of PgT relates to the high measurement accuracy of the qualifying genetic biomarker and its relationship to the drug target.24The practical advantages of PgT include improved treatment efficacy, avoidance of unnecessarily treating non-responders, decreased clinical trial sample size requirements, improved pharmacoeconomics, a better safety profile, and improved commercialization potential.24These advantages have been particularly successfully applied in oncology, where tumor somatic cell mutations can be relatively readily identified and specific pharmacologic therapy devised. However, with germline genetic variation progress has been slow, and there is still not an approved PgT cardiovascular drug.

[0213] If the described clinical trial is positive, and in particular if supported by the additional pharmacogenetic and mechanistic data from this proposal, bucindolol (Gencaro™) will be the first approved PgT cardiovascular drug. Moreover, PgT target expansion and results from the proposed basic mechanism experiments may encourage the pharmaceutical industry to intensify precision / PgT drug development for other cardiovascular indications.Increased Adrenergic Signaling in Heart Failure and Atrial Fibrillation

[0214] Although human cardiac myocytes contain 3 types of B-ARs, Bl-ARs dominate and are thought to mediate most pathologic effects of chronic adrenergic stimulation in the human failing heart.26Increased cardiac adrenergic drive signaling through Bi-ARs is responsible for an important component of pathologic eccentric remodeling in HFrEF patients, the inhibition of which explains the beneficial effects of B-blockers.1,26,27The sustained increased in cardiac adrenergic drive in HF and AF is due to increased sympathetic neuronal release and decreased re-uptake of norepinephrine (NE),28for which the Bl-AR has high affinity.26Although sustained increased cardiac adrenergic drive has a net myopathic effect on the myocardium26including predisposing to arrhythmias,29evidence indicates that certain cellular mechanismscounter these adverse effects. The most obvious are B-AR desensitization and downregulation that diminish adrenergic signaling.30However, desensitization also includes signaling a pathway that actively counters the adverse effects of sustained adrenergic drive, as discussed in Sections 1.5 and 1.6.

[0215] As subsequently discussed, in certain B-AR genetic variants, this pathway may be therapeutically activated by bucindolol, leading to improved efficacy and expansion of the PgT target population.Bucindolol Pharmacology and Pharmacogenetics.

[0216] B-blockers are classified by their perceived advantageous pharmacologic characteristics prevailing during the generation of development.31Bucindolol, which blocks both Bl- and B2-ARs with high (low nanomolar KD) affinity, was originally classified as a "3rd generation" compound based on its mild vasodilator activity that is likely due to weak ocl-AR blockade.3233When it entered PgT development bucindolol was re-classified as a 4th generation compound.26The primary receptor gene variant currently targeted by bucindolol is shown in FIG. 38, the protein product of the major allele of the codon / amino acid position 389 Bl- AR.polymorphism. In the intracellular domain, a nonsynonymous SNP (cytosine -> guanosine transversion) leads to an arginine (Arg) being replaced by a glycine (Gly),34resulting in a reduction in function. The ADRB1 Arg389Gly polymorphism has a minor allele frequency (MAF) that ranges from 0.28 for European ancestry (EA) to 0.43 in African ancestry (AA)35and thus is relatively common. As a result only approximately one- half the U.S. population is homozygous for the Arg389 genotype. Compared to Arg389, the Gly389 receptor exhibits a 3- 4 fold loss of signal transduction function,21 ,34,and fewer constitutively active receptors.36Bucindolol is an inverse agonist (inactivates active state receptors) for Arg389 but not for Gly389 receptors,21likely contributing to its greater efficacy in patients who are homozygous for Arg389. The Gly389 variant also has lower binding affinity for NE compared to Arg389, with ADRB1 Arg389 homozygous genotype nonfailing hearts having 3 to 7- fold more receptors in a high affinity NE binding state.37The importance of higher NE affinity for the Arg389 Bl -AR is that bucindolol, uniquely among B-blockers, has sympatholytic properties (lowers NE) in HF38and AF / HF19patients. NE lowering also contributes to its selective efficacy in patients with 389Arg vs. Gly389 Bl-ARs, by reducing high affinity NE signaling.

[0217] The second nonsynonymous SNP that affects receptor biology is in the extracellular domain at codon 49, an adenosine to guanosine transition resulting in a serine (Ser) being replaced by a Gly (ADRBI Ser49Gly). This polymorphism doesn’t affect receptor signal transduction or binding affinity, but has major effects on receptor internalization and desensitization from agonist exposure, with Gly49 readily internalizing and Ser49 being internalization resistant.39,40The codon 49 and 389 polymorphisms dictate differences in gene haplotypes, creating the potential for both disease modification and effects on pharmacologic therapy. For example, when occupied by NE an Arg389Gly haplotype Bl-AR would be expected to deliver a high level of signal transduction to a receptor that easily internalizes. In contrast, an Arg389Ser49 haplotype would produce a receptor that has a low probability of internalizing and therefore would not have access to cardioprotective pathways (Section 1.6). Bucindolol32,33,41as a “biased ligand”42is similar to but more potent than the biased ligand carvedilol41,43,for inducing Bl-AR behavior that leads to receptor internalization (as deduced from receptor down-regulation / sequestration without a decrease in mRNA in chick heart cells),41and it is a higher affinity Bi-blocker.33One of the main reasons bucindolol was originally developed for HF treatment was that it is a competitive antagonist of both Bl- and B2-ARs, with respective average KiS across multiple methods of analysis of 3.6 and 5.0 nM compared to carvedilol’ s of 4.0 and 29.1 nM.33B2-ARs are present on human cardiac myocytes and coupled to many of the same pathways as Bl,44albeit with some nuances.45Human failing left ventricles (LVs) have a Bl :B2- AR ratio of - 65:35 compared to 80:20 in nonfailing hearts,44due to selective downregulation in Bl-ARs. Although B2-AR signaling is inherently less myopathic than Bl,26at high expression levels B2-ARs produce a cardiomyopathy.46,47There is also evidence that B2-AR signaling is more arrhythmogenic than Bl.48The original idea behind both bucindolol and carvedilol as treatments for HF was that, based on their vasodilator properties, they would inhibit the substantial number of B2-ARs in the failing EV without precipitating excessive worsening of HF.49Although both bucindolol and carvedilol proved to be well tolerated in Class I-III HF, an attempt in the Phase 3 NIH-sponsored BEST trial to extend bucindolol into very advanced HF, including patients that were fluid overloaded and unstable, compromised efficacy results.50,51However, in stable HFrEF patients with the same EVEF and other criteria the clinical results with bucindolol and carvedilol are essentially identical,26with bucindolol’ s efficacy being concentrated in patients with the ADRBI Arg389Arg genotype where its results in both HF and AF / HF are superior to carvedilol’ s or metoprolol.19,21,52,53Bl- and 2- adrenergic Receptor Biased Ligand Signaling

[0218] Biased ligand signaling can be defined as “a ligand that binds to a receptor and signals to a variable extend through both G protein-dependent and protein-independent pathways”,42or alternatively “ligands acting on the same receptor resulting in disproportional induction of different signaling responses”.54Bucindolol is a biased ligand for human Bl- and B2-ARs by virtue of engaging both the canonical adenylate cyclase / cAMP and the non- canonical MAP kinase ERK1 / 2 pathways.42,43,55,56These properties are similar to carvedilol’ s,57which is FDA approved for the treatment of HFrEF and hypertension. Each compound uniquely binds to the same regions in B-ARs, distinct from other B-blockers.42Despite their biased ligand similarities the two compounds have important differences; carvedilol doesn’t lower NE or exhibit Bl-AR Arg389 inverse agonism in isolated human heart preparations,21and has no enhanced efficacy in ADRB1 Arg389Arg vs. Gly389 genotypes.52,53Although both exhibit biased ligand behavior and G-protein independent Src kinase / EGRF / MAP kinase / ERKl / 2 signaling,43,55-57carvedilol apparently (there are countervailing data5458’) signal transduces this pathway through B-arrestins,58while the non G-protein signal transduction mechanism for bucindolol hasn’t been identified.55

[0219] The approach used herein to the unresolved issue of what signal transduces the MAP kinase ERK1 / 2 pathway, as well as other further details of bucindolol’ s biased signaling, is to perform these studies in human cardiac-based systems.The Importance of Cell Surface Membrane Internalization in Signaling the Src kinase / EGFR / MAP Kinase ERK1 / 2 pathway.

[0220] For most G-protein coupled receptors (GPCRs) including Bl- and B2-ARs, sustained (minutes to hours) occupancy by an agonist and subsequent phosphorylation by G- protein receptor kinases (GRKs) leads to “internalization” of the agonist-bound receptor via binding of B-arrestinl and 2 to the phosphorylated receptor. B-arrestins also undergo a second binding event where they intercalate into the receptor core and compete for coupling to Gs, resulting in desensitization. Internalization is an endocytic process resulting in a ligand-bound receptor moving from the outer plasma membrane to the inner portion into endosomes,59which then traffic to the cytosol. For both Bl-60and B2- ARs57internalization serves as a bridge between the rapid uncoupling via GRK phosphorylation / B-arrestin binding and downregulation, the net loss of cellular receptors. Internalized receptors are also a “depot” thatcan be either recycled to the cell surface, degraded (leading to downregulation), or in some cases activated if the cell membrane is permeable or in contact with endocytic vesicles. B- arrestin is a scaffolding protein,60-62and not only assembles the necessary components for internalization,62,63but also is required for stimulation of non-G protein mediated MAP kinase (ERK1 / 2) signaling and EGRF transactivation.60For both Bl-60and B2-ARs57internalization of the agonist-receptor complex is required for ERK1 / 2 pathway activation. This pathway is cardioprotective via mechanisms such as inhibiting apoptosis, and in model systems its activation attenuates the development of catecholamine cardiomyopathy.60,61The common entity between receptor uncoupling from G protein, initiation of downregulation via internalization, and non-canonical signaling is B-arrestin. Therefore, receptor recruitment of B- arrestin and the subsequent signaling events must be measured to account for the effects of biased agonists, in addition to measuring the canonical, (Gs / cAMP) component.

[0221] In addition to agonists, a variety of antagonists are capable of initiating internalization and / or B-arrestin recruitment.64-67For Bl-ARs the antagonists carvedilol,57,61nebivolol,68and by proxy evidence bucindolol33,41-43,are associated with one or more B- arrestin-mediated events such as downregulation without an mRNA decrease,41or MAP kinase ERK1 / 2 activation.45,43,55,56However, internalization of Bl-ARs has not been directly evaluated for bucindolol, as only relatively longer term (24 hour) exposure has been investigated and in avian Bl-ARs41that display atypical receptor sorting.72,69,70Nevertheless, bucindolol is abiased ligand42that activates the Src kinase / EGFR / / ERKl / 2 pathway via both Bl- and B2-AR signaling,43,55without increasing cAMP levels in model system B2-ARs41or human cardiac preparations32,33(i.e., biasing towards B-arrestin and away from Gs).Identification of Additional Pharmacogenetic Targets for Bucindolol.

[0222] In HFrEF patients bucindolol has a selective inhibitory effect on the Arg389 Bl -AR that exceeds standard B-blockers for HF endpoints21,37,or reduction in AF burden (AFB).19However, bucindolol’ s "pluridimensionality of efficacy"43,55or "ligand biased signaling",42has not been exploited for efficacy enhancement. As presented in herein, expansion of the PgT targeted population to include ADRB1 Arg or Gly389Ser49 haplotypes or they in combination with non-internalizing ADRB2 haplotypes will increase the eligible target population, in addition to enhancing treatment effect.

[0223] Bucindolol was originally developed as a nonselective P-blocker that, unlike propranolol, was well tolerated by HFrEF patients.3171The impetus for developing nonselective [3-b lockers for the treatment of heart failure was that 35-40% of the P-adrenergic receptors in the failing heart are [32, coupled to canonical cAMP signaling and a positive inotropic effect similar to (31-ARs.43,46Although sustained increased [32- AR signaling is less cardiomyopathic than [ 1, it is associated with myocardial damage and pathologic hypertrophy.46,47Primarily based on similar treatment effects including favorable effects on mortality between pi-AR selective and nonselective pi / p2-blocking agents26the current dogma is that myocardial [32- AR signaling and its inhibition is not important in therapeutic inhibition of sustained hyperadrenergic drive in heart failure. However, data contained in Figures. 8-15 summarized in Figures 5-7 suggest this is not the case. When heart failure patients with non-internalizing P2-AR haplotypes are treated with the biased ligand bucindolol, for both the ACM / Tx an AF / AFL / ACM endpoints the treatment effects are additive to ADRB1 Arg389Arg and Gly389 carriers, as well as to the Arg389Ser49haplotype (FIG. 42). In the higher, >20% LVEF cohort the additivity to either Arg389 or Gly389 genotypes or haplotypes is even more pronounced. Furthermore, K-M curve data in Figures 8-15 demonstrate a consistent difference between the treatment effects of bucindolol in favor of non-internalizing ADRB2 haplotypes (all haplotypes except Gln27Argl6), with attenuation or abolishment of treatment effects when 1 or 2 copies of Gln27Argl6 are present.

[0224] Whereas for ADRB1 a Ser vs. Gly at position 49 determines the internalization response to agonists39,40and based on data in this application to the biased ligand bucindolol, for ADRB2 internalization it is haplotype structure that influences internalization.72,73In Chjnese Hamster fibroblasts the Gln27Argl6 haplotype undergoes rapid downregulation and increased intemalization / sequestration with isoproterenol, and when Gln27 is mutated to Glu the expressed receptors are resistant to downregulation and internalization.72Due to linkage disequilibrium the Glu27Argl6 haplotype is not present in most genetic studies,73including the BEST Adrenergic Receptor Polymorphism substudy analyzed in this application. However, in a human investigation of rapid desensitization of vascular responses to isoproterenol infusion, only the Gln27Argl6 haplotype was associated with rapid desensitization,73a proxy for internalization. In the application the inventors used any copy number of Gln27Argl6 to denote internalization, and designated all other haplotypes as noninternalizing. The non-internalizing haplotypes include Glu27Glyl6 homozygotes,Glu27Glyl6 / Gln27Glyl6 heterozygotes, and Gln27Glyl6 homozygotes. In ADRB1 Arg389Arg subjects 80% of the "Not Gln27Argl6" haplotypes contain a Glu27, and in Gly carriers 74% have at least Icopy of Glu27. Thus a Glu 27 genotype is a partial proxy for noninternalizing, Not Gln27Argl6 haplotypes. The data in Figures 8-14 summarized in Figures 5-8 indicate that these assignments are correct.Haplotype identification.

[0225] Historically, single allele measurement of haplotypes has been technically challenging, resulting in slow turnaround and relatively high costs. As a result most haplotype data are generated by genotype imputation, which is approximately 95% accurate74and whose accuracy increases when strong linkage disequilibrium eliminates a haplotype. To generate haplotype preliminary data in Section 4 the inventors used such a method, the “Haplo. stats” package available in R.75Fortunately, the recent development of bar-coded circularized amplicons and "Long Read" sequencing76has resulted in haplotype identification that is high throughput, economical, and highly (99.9%) accurate. Aspects herein use Pacific Biosciences version of the Long Read methodology to measure haplotypes,77with the bar-coded amplicons and sequencing performed at the BYU DNA Sequencing Center. This methodology will be selectively used for resolution of ADRB1 and ADRB2 haplotype heterozygotes in the planned clinical trial, to provide regulatory certainty for haplotype labeling in prescribing information.Novel Primary Endpoint for the Phase 3 AF / HF Trial.

[0226] In the Phase 3 AF / HF trial (PRECIS ION-AF) the inventors will deploy a primary endpoint developed with the assistance of FDA's Clinical Outcomes Assessment Division. The validation and the results comparing endpoint efficiency to standard endpoints are submitted for publication. The endpoint is “Symptoms Burden of AF” (SxBAF), and based on comparative power it is at least 3x more efficient than the traditionally used time to first symptomatic AF event endpoint. The impetus behind the development of SxBAF was that bucindolol's biologic mechanism of action is time dependent, and any evaluation of AF prevention efficacy should extend over the entire period of follow-up. It can be observed in FIG. 40B that the separation of the bucindolol-metoprolol succinate curves for the AFB (2A) or SxBAF (2B) begins at 4-6 weeks, followed by an increasing treatment effect thereafter. The SxBAF plot is from the submitted paper, using symptoms of AF N=5) and HF (N=5) measured by a Patient Reported Outcomes (PRO) instrument, the AFSQ,78FDA does not currentlyrecognize AFB as a registrational primary endpoint, but they will accept symptoms aligned to an ECG documented AF episode such as in SxBAF, which is the first method developed to measure the entire burden of AF symptoms. With SxBAF, trial size can be reduced by up to 50% while actually increasing statistical power. The Phase 3 PRECIS ION- AF trial is planned for an efficacy cohort of 300 subjects, plus an additional supplementary group of 100 subjects with a combination genotype of {ADRB1 389Gly carrier + Ser49Ser} (Section 4).Measurement of Gene Expression in the Intact Heart.

[0227] The Bristow Eaboratory and collaborators were the first to measure gene expression in the intact human heart, using interventricular septum EmBx material for RNA extraction and proprietary PCR-based technology for determination of mRNA abundance.79This was used to demonstrate that the "biologic effects"27of B-blockers are due to favorable changes in the expression of candidate genes whose encoded proteins mediate pathologic hypertrophy and systolic dysfunction.80The preferred design is a serial study,1,8°’83with baseline measurements prior to an intervention and one80or two1,80-83follow-up measurements, in order that participants are investigated on their own genetic and other backgrounds. The latest study revealed that the Bl -AR is connected to a downstream network of at least 430 genes1that regulate eccentric pathologic remodeling (a dilated LV with systolic dysfunction). Reversal of these genes’ expression by B-blocking agents likely accounts for their reverse remodeling effects, which are associated with the largest reduction in mortality of any HFrEF drug therapy.1,26This study also implicated long noncoding RNAs (IncRNA) in the regulation of the Bl -AR gene network,1joining microRNAs (miRs)2as having a regulatory role. The current proposal describes the use of RNA from IVS EmBxs to track changes associated with EV and LA reverse remodeling, since these chambers are in series and are subjected to the same degree of changes in wall stress, a big determinant of remodeling gene expression changes.84In addition, the inventors are adding to the usual bulk RNA analyses measurement of a subset of genes in cardiac myocyte rich areas devoid of any other cells.Human Induced Pluripotent Stem Cell Cardiac Myocytes (iPSC-CMs).

[0228] Comparison of IPSC-CM to myocardial gene expression in the same subjects. The inventors worked a project where patients had their IVS gene expression data compared to that from their PBMC origin iPSC-CMs. This project was begun as a feasibility exercise towards the end of PROBE-IT, and only enrolled 2 patients. However, it did demonstratefeasibility, and the inventors plan to perform it in the PRECISION-AF substudy. This will be the first study of its kind, i.e. a comparison of iPSC-CM gene expression to that in the same subject's intact heart.

[0229] Biased signaling in iPSC-CM ADRB1 389 / 49 haplotypes. In a previous publication81using a CRISPR editing approach, one group introduced and corrected LAMP2 gene variants in human iPSC-CMs from Danon cardiomyopathy patients. In aspects herein, iPSCs will be gene edited by CRISPR85,86to the 4 ADRB1 or ADRB2 haplotypes, including the Gly389 / Gly49 haplotype that is rarely observed in genetic studies. These haplotypes have not been previously gene edited or investigated in iPSC-CMs. The Gs / canonical vs. B- arrestin / ERKl / 2 pathway protocol that is being run in HEK293 cells and isolated human heart preparations will be used in gene edited iPSC-CMs.\

[0230] The general approach is to perform most studies in patients or human derived cardiac material, using state of the art methodology and study designs.PRECISION-AF Trial and Interface with this Proposal.

[0231] The PRECISION-AF trial is a 400 patient Phase 3 trial designed to gain approval of bucindolol for prevention of AF in AF / HF patients. The primary endpoint is SxBAF, and the 300 patient primary efficacy cohort consists of HF subjects with an ADRB1 Arg389 homozygous genotype, a pre-randomization EVEF of 0.36 to 0.54, and at least 1 symptomatic AF episode in the past 4 months. The control for bucindolol is metoprolol succinate (Toprol- XE) as used in Phase 2,87with a trial duration of 180 days. Aspects herein include an additional 100 patients with ADRB1 Gly389 and Ser49 carrier genotypes, designed to deliver sufficient numbers of Gly389 / Ser49 haplotypes for evaluation. At the end of the ADRB1 Arg389Arg genotype 300 patient trial, if the Gly389 / Ser49 haplotype data are positive / in agreement with the preliminary data described in Section 4, the Phase 2 100 patient trial will convert to Phase 3 in a seamless trial design,87enrolling patients with 2 copies of the ADRB1 Gly389 / Ser49 haplotype (FIG. 40). PRECISION-AF is expected to be completed in 3 years, with an additional 6 months to finish data analysis. Study initiation is planned for QI 2025.

[0232] Left Atrial and Left Ventricular Remodeling. The primary imaging method used to measure FA and LV remodeling will be 3D (for the LV) and 2D (FA) echocardiography. Cardiac MRI will also be performed and will use late gadolinium enhancement as a measure of fibrosis in both the EV and LA. CMR LA and LV volume and strain measurements will beused as primary data if the echocardiographic images are suboptimal. Subjects will undergo a standard 2D and Doppler echocardiogram at baseline, 12 months and 24 months including 2D Biplane EF and Doppler indices as recommended by the 2016 American Society of Echocardiography guidelines. Speckle tracking imaging will be used for LA and LV volumes and strain. LA strain will focus on the reservoir aspect; however, the conduit and contractile or boost functions will also be measured.88CMR including high resolution late gadolinium enhancement (LGE)89will be performed as previously described,90in the UCH imaging center. The primary endpoint for LV remodeling will be the ejection fraction,1and for LA it will be strain imaging by 2D-echo with CMR91as a back-up. In order to achieve 80% power on the expected changes, based on 50% of what was achieved at 3 months in a previous B-blocker LV remodeling study with bucindolol92an 80 patient sample size (with 10% attrition) is required. Consequently, 40 additional patients for echocardiography measurements will be recruited from a planned Remodeling Substudy in PRECIS ION-AF, supported by the sponsor.

[0233] Myocardial Gene Expression. These studies will consist of candidate and global gene expression (mRNA, IncRNA, miR) measurements by RNA-sequencing as previously described,1using RNA extracted from IVS EmBxs.90For safety reasons it is not feasible to obtain LA tissue by EmBx, but for selected genes including ion channels LV gene expression may be a surrogate (Section 3.3). Focal gene expression in cardiac myocyte enriched regions will also be measured, by the 10X Genomics-Visium technique93in a single biopsy fixed for this purpose. Gene expression will be measured at baseline just prior to randomization, and at end of study at 24 weeks- 180 days. Changes in candidate and global gene expression will be compared to LA and LV remodeling indices by previously described methods.1

[0234] Canonical vs. MAP kinase ERK1 / 2 Signaling Pathway Protocol. The protocol for investigating the chain of events of bucindolol’ s activation of MAP kinase ERK1 / 2 will be applied to all models (HEK293 cells stably expressing transfected Pi- and 2-AR haplotypes, gene edited iPSC-CMs expressing these haplotypes, iPSC-CMs generated from subjects enrolled in the clinical trial, and isolated RA and RV preparations from explanted human hearts). HEK293 cells and iPSC-CMs will be utilized to dissect the molecular mechanisms of differential responses to bucindolol of the different B-AR haplotypes. HEK293 cells have the advantage of being readily transfected and amenable to altering the stoichiometry of receptors, B-arrestins, effectors and high-throughput assays. iPSC-CMs are the cell type of interest possessing physiology relevant to myocyte contractility, arrhythmogenesis and ion channelfunction. For measuring cAMP (representing Gs coupling) a standard plate-based assay is utilized as previously reported.94B-arrestin recruitment in HEK293 cells is measured as previously published.95Receptor internalization is measured by cell surface / whole cell radioligand binding as previously published.95Multiple time points of bucindolol or NE (the positive control) are used to calculate response and maximum. For ERK1 / 2 activation, immunoblots with specific antibodies for total and phosphorylated ERK1 / 2 are used. Exposures to bucindolol or agonist (NE with Bl-ARs and the counterpart B-AR blocked) are performed in the absence and presence of a PKA inhibitor to isolate B-arrestin-mediated (vs. Gs / PKA-mediated) activation. Src-kinase activity will be measured by pharmacologic inhibition by PP260and the more selective inhibitor saracatinib.96For iPSC-CMs a different assay to detect B-arrestin recruitment will be used, since those cells will have been gene-edited to express the various receptor haplotypes without tags. This assay, termed “bystander BRET”,55requires that the cells be transfected with a cell membrane expressed tagged protein. Since functional B-ARs are intercalated in the cell membrane, recruitment of tagged B-arrestin to the receptor is readily detected by complementation with the membrane protein.55The Liggett Laboratory uses GFP fused to the fatty acylation motif of Lyn-kinase that is plasma membrane bound, and RLuc2 fused to B-arrestin. The readout is a bioluminescence energy transfer (BRET) signal that increases in an agonist- dependent manner as B-arrestin is redistributed from cytosol to the membrane-bound receptors.

[0235] PSC-CMs. Peripheral blood mononuclear cells (PBMCs) isolated from 5 ml of human blood will be reprogrammed into iPSCs using a Sendai reprogramming kit (CytoTune™-iPSC 2.1, ThermoFisher Scientific). After validation of karyotyping and pluripotency, human iPSC lines will be induced to differentiate into iPSC-CMs expressing atrial or ventricular phenotype as detected by action potential, as described inOL11-20-86-97-98and others99publications. Immature iPSC-CMs generally express low levels of adrenergic receptors. However, human iPSC-CMs cultured using an established protocol exhibit structural and functional maturity, increased ADRB1 expression, and response to B-adrenergic agonist treatment20,86(FIG. 41 A). These mature IPSC-CMs also respond to activation of MAP kinase ERK1 / 2, as shown in Figure. 4B.20

[0236] Global gene expression and fil-AR haplotype measurements. In PRECISION-AF study-subject specific iPSC-CMs global gene expression will be measured and compared to myocardial gene expression measurements performed by the same RNA-Seq methodology. Forthese experiments PBMCs will be collected from patients entering the PRECISION-AF trial and AAI substudy at CU-AMC, frozen and shipped for processing. Mature iPSC-CMs will be generated as described herein, and some of them will be gene edited to the 4 haplotypes for both Bl - and B2-ARs. Both these and the study subject specific iPSCs will be used for protocols disclosed herein. In addition, RNA will be extracted from iPSC-CM aliquots (both haplotype edited and subject-specific) and shipped frozen for global gene expression measurements, where results will be analyzed by haplotype and compared to myocardial gene expression in EmBx material taken from the same subject. Haplotypes will be measured.

[0237] Physiologic Measurements. Human iPSC-CM engineered to carry the 4 ADRB1 or ADRB2 haplotypes will be seeded into fibronectin coated 24-well MEA plates (Cytoview, Axion Biosystems) at density of 100,000 cells per well and cultured in myocyte media for 2 weeks before recording with MaestroEdge MEA system (Axion Biosystems).20The following measurements will be obtained in spontaneously beating monolayer of iPSC-CMs: Contractility, beating rate, extracellular field potentials, excitation-contraction coupling, and waveform propagation are all measured by the MEA system. These parameters will be recorded before and after pharmacologic manipulations that include: NE (0.1 uM), in the presence of a- AR (prazosin luM) and B2- AR (ICI 118,551, 0.3 uM) antagonists; bucindolol (0.1 uM); or metoprolol (0.3 uM). This protocol will be conducted in both atrial and ventricular phenotype CMs.For study-subject specific measurements carried out in pre -randomization iPSC-CMs the same protocol will be applied, in concert with their having global gene expression and B-AR haplotypes measured.

[0238] Isolated Human Heart Preparations. Isolated human heart preparations treated with NE, bucindolol or metoprolol will be investigated for 1) internalization of Bl- and B2- ARs, and 2) activation of MAP kinase ERK1 / 2. RV trabeculae from explanted end stage failing hearts removed from cardiac transplant recipients or nonfailing hearts from unused organ donors will be mounted in a 16 chamber tissue bath and paced at 1 Hz as previously described.21Right ventricular trabeculae and right atrial (RA) strips100(on explanted failing hearts there typically aren’t sufficient LA trabeculae for isolated tissue studies) removed from the same explanted heart will be investigated, under the same conditions. Each experimental group will be assigned 2-3 tissue baths, for the evaluation of 5-6 different conditions. To investigate receptor- ligand internalization of Bl-ARs, 5 minute and 1 hour incubations will be carried out with: L-NE 0.1 uM + a- AR (prazosin) and uptake- 1 (desipramine) blockade; bucindolol HC10.1 uM; metoprolol tartrate 0.3 uM; and vehicle controls. Membrane preparations will be assayed for Bl-ARs by radioligand binding using125[I]CYP to label the total B-AR pool, with competition binding by selective antagonists according to previously described methods.44,47.Two types of membrane fractions will be assessed, a heavier weight fraction consisting of plasma membrane bound receptors and a lightweight fraction containing internalized receptors recovered from a sucrose gradient and subjected to high speed centrifugation of the heavy membrane supernatant.44Phosphorylated and unphosphorylated ERK1 / 2 will be measured as described in Section 3.4. Assuming phosphorylated ERK1 / 2 is identified after ligand incubation in some groups, the protocol will be repeated in the presence of Bl- or B2-AR blockade to identify the receptor pathway responsible.

[0239] Statistical Considerations. The clinical trial primary endpoint of SXBAF will be analyzed by multi-level negative binomial regression as shown in FIG. 39B, gene expression data are analyzed by non-parametric methods described in references (1) and (85), and A P value of <0.01 is required by FDA for the clinical trial primary endpoint, and otherwise a P <0.05 with a Benjamini-Hochberg correction for multiplicity will be taken as statistical significance.Functional Importance of B- adrenergic Receptor Polymorphisms and Relationship to Bucindolol

[0240] ADRB1 Arg389Gly. The human Bl-AR is the product of an intronless gene, encoding a 477 amino acid protein34(FIG. 38). As first reported by the Eiggett Laboratory, the encoded36mRNA is highly polymorphic, with 26 total SNPs of which 13 are nonsynonymous. Of these 13 amino acid changes 2 result in effects on receptor function or trafficking that form the basis of PgT targeting of the B-blocker bucindolol (Section 1.4.2, FIG. 38). The evidence that the Bl-AR 389 Arg / Gly polymorphism can affect the response to B-blocking agents was generated in the NIH sponsored substudy of the BEST trial "Pharmacogenomics of Beta- Adrenergic Receptor Polymorphisms and Response to Beta-Blockers in Heart Failure" performed in the BEST trial,21. The results of the BEST Adrenergic Receptor Polymorphism substudy demonstrated that, compared to the counterpart genotype of Gly389 carriers, patients who were ADRB1 Arg389 homozygotes had a much greater treatment benefit for all-cause mortality (ACM) and other HF endpoints,21,37as well as prevention of AF19or VT / VF / sudden cardiac death.101It was initially assumed that the differentiation of treatment effects between Arg389 and Gly389 carriers would apply to all B-blockers, but work done in isolated humanheart preparations in the Bristow Laboratory provided evidence that the PgT effects were specific to bucindolol, based on its inverse agonism for Bl-Arg389 ARs21and its unique NE lowering properties.38The BEST parent trial was stopped short of a statistically significant effect on prevention of mortality (P=0.053),26with the failure to produce a larger mortality reduction ultimately shown to be due to enrolling patients whose HF was too advanced (NYHA Class IV51and fluid overloaded Class III);26this type of patient is no longer enrolled in B- blocker trials. Ultimately because of the size and cost of performing a PgT-based Phase 3 trial in HF, ARC A biopharma decided to develop bucindolol for prevention of AF in HF patients, in part because PgT differentiation of bucindolol for Arg389 vs. Gly389 is even more obvious for arrhythmia endpoints.22,101TABLE 2-*By unadjusted Cox modeling; AF+=AF / AFL / ACM, subjects not in AF at randomization;ACM=al 1-cause mortality, AF=atrial fibrillation, AFL=atrial flutter; C VM / HFH=combincd endpoint of time to CV mortality or HF hospitalization, C VH=cardiovascular hospitalization

[0241] ADRB1 Ser49Gly and ADRB2 Gln27Glu. The ADRB1 position 49 Ser / Gly polymorphism was also investigated in the BEST Substudy, with major / minor (Ser / Gly) allele frequencies reported as 0.86 / 0.14 in EA and 0.76 / 0.24 in AA35In model systems from the previous studiesthe Gly variant exhibits markedly greater receptor internalization and / or downregulation in response to agonists.41,42,102As detailed herein, agonist induced internalization of Bl- and B2-ARs is linked to cardioprotective MAP kinase ERK1 / 2 signaling. This is a potential reason why Bl -AR Gly49 genotypes and the Arg389Gly49 haplotype are associated with less ventricular fibrillation (VF) during myocardial infarction, as recently reported by the Bristow laboratory working with the MAP-MI clinical trials group.103In addition, in the MAP-MI study ADRB2 Gln27 genotypes, encoding a receptor prone to agonist- induced desensitization / internalization,72,73in combination with ADRB1 Gly49 genotypes was also protective of VF.103In contrast, the ADRB2 Glu27 variant is relatively resistant to desensitization and downregulation.72,73,104These data contributed to the formulation of the hypothesis tested in Aims 3 and 4, i.e. that on exposure to agonists or non-agonist biased ligands, internalized Bl- and potentially B2-ARs are cardioprotective for arrhythmias as well as worsening HF. The internalization-resistant variants for both ADRB1 (Ser49) and ADRB2 (haplotypes other than Gln27Argl6) have relatively high allele frequencies, 0.86 (EA) or 0.76% (AA) and 0.39 (EA or 0.19 (AA), respectively.35This means that if a biased ligand could promote internalization and MAP kinase ERK1 / 2 signaling in individuals with these variants there would be potential for a therapeutic effect. Table 2 (genotypes) and FIG. 42 (haplotypes), from a subpopulation of the BEST polymorphism substudy that most closely resembles entry criteria in the planned Phase 3 trial, (LVEF >0.20), contain data relevant to this hypothesis. For Gly389 carrier combination genotypes (Table 2, Rows 1-4), with the addition of noninternalizing receptor alleles (Rows 2-4) there are declining hazard ratios (HzRs, ((l-HzR)xlOO = treatment effect) for all endpoints but CVM / HFH. In row 4 where 2 non-internalizing genotypes are incorporated, ACM reaches statistical significance with a HzR of 0.43, P = 0.045, and across all endpoints the HzRs approach the range of Arg389Arg (Row 5). For Arg389Arg subjects, with the addition of non-internalizing variants a generally declining HzR is also observed. These patterns are all statistically significant compared to the counterpart internalizing genotype combinations, and a binomial test for departure from random occurrence was significant at a P = 0.0001 across 8 endpoints.

[0242] [J-AR Haplotype Clinical Outcomes Data. Figures 5A and 5B give the effects of Bl- and B2-AR haplotypes on bucindolol vs. placebo treatment effects by 1 or 2 copies (i.e.heterozygous or homozygous) of the alleles, for the time to all-cause mortality or cardiac transplantation (ACM / Tx) and AF / AFL / ACM endpoints in the same patient population as in Table 2. ACM / Tx was selected because it was the primary endpoint in the BEST adrenergic receptor polymorphism substudy, and time to AF, AFL or ACM was the primary endpoint in the GENETIC- AF trial.78,87For ACM / Tx and ADRB1 Arg389 haplotypes in the entire cohort (LVEFs range 5% to , 40%, 1 or 2 copies of the “internalizing” Arg389Gly49 haplotype (FIG. 42A, section D rows 1 and 2) demonstrate no difference in event rate between the Bucindolol (B) and Placebo groups and a nonsignificant hazard ratio (HzR, from Kaplan-Meier curves) for either 1 or 2 copies of the allele. However, with 2 copies of Arg389Gly49 the Placebo event rate is lower than for any of the non-internalizing haplotypes descending down the column (Sections E and F, rowsl and 2). For the AF / AFL / ACM endpoint in the entire LVEF cohort (FIG. 42A) the same pattern of a lower event rate with 2 copies of Arg389Gly49 compared to 1 copy plus equivalent event rates in the placebo and bucindolol arms is seen. Similar results are seen for the LVEF >20% cohort (FIG. 42B). Declining event rates per copy number in the placebo group resulting rates not different from bucindolol suggests that the self-internalizing Arg389Gly haplotype is having a beneficial effect, comparable to that of bucindolol and resulting in nonsignificant HzRs. Due to linkage disequilibrium the Gly389Gly49 haplotype was not present in this and rmost other b-AR genetic studies. With the ADRB1 Arg389Ser49 haplotype, progressing from 1 to 2 copies and then adding the "Not Gln27Argl6" haplotypes that include Glu27 constructs as well Gln27Glyl6, there is an improving bucindolol treatment effect, for ACM / Tx culminating in HzRs of 0.27 (P=0.020) and infinitely low due to no deaths in the bucindolol group in the entire LVEF and LVEF >20% cohorts respectively (Section E, Tables 5A and 5B). The AF / AFL / ACM endpoint yields a similar pattern, in both cohorts. For both endpoints in both cohorts there is no change in the Placebo event rate as the number of non-internalizing haplotypes increases. For Gly389Ser49 haplotypes and ACM / Tx Figures 5A and 5B, Section F) the decreasing HzR / ascending treatment effect is similar to that in Arg389Ser. The AF / AFL / ACM endpoint (Figures 5 A and 5B, Section F) exhibits a dose response from 1 to2 copies of Gly389Ser49, but unlike for ACM / Tx the addition of the noninternalizing ADRB2 haplotypes has no effect.

[0243] Possible target expansion based on haplotypes is apparent for ADRB1 Gly389 genotypes that are currently excluded from treatment. Using a criterion that treatment eligibility of a Gly389 haplotype should be associated with a treatment effect (1 -hazard ratio) >90% of that for Arg389Arg subjects averaged across both endpoints in FIG. 42, in the entire cohort(FIG. 43) the 2 copy Gly389Ser49 haplotype achieves average relative increases in treatment eligibility of 19% in the entire cohort (from 47.4% in Arg389Arg to 56.3%, FIG. 43) and 17% in the LVEF >20% cohort (from 45.3% to 54.4%). The 2 copy Gly389Ser49 haplotype was present in 17.0% of the Gly389 and 8.9% of the entire cohort patients represented in FIG. 42A. For the LVEF >20% cohort (FIG. 42B) the respective percentages are 16.8 and 9.2. Adding Not Gln27Argl6 haplotypes to 2 copies of Gly389Ser49 yields a further lowering of the ACM / Tx HzR and increase in treatment effect for ACM / Tx, in both cohorts. Furthermore, in the LVEF >20% cohort (FIG. 44) Not Gln27Argl6 haplotypes added to Gly389 carrier subjects increases the average treatment effect from 28% to 49%, on par with the Arg389Arg treatment effect of 48%. This provides a 43.2% relative increase in pharmacogenetically eligible patients, and when added to boost given by the addition of Gly389Ser 49 subjects the relative increase in eligible subjects is by 57% (from 45.3% in Arg389Arg to 70.9%) by For efficacy enhancement by ADRB1 Arg389 / Ser49 and then the ADRB2 Not Gln27Argl6 haplotypes ,in comparison to Arg389Arg subjects the number of subjects with pharmacogenetically enhanced responses (to the level of Arg389Arg in Gly carrier subjects or to <Arg389Arg these subjects) is increased in the entire cohort from 47% (493 / 1040) to 72% (74 / 1040) (FIG. 43), and in the LVEF >20% cohort from 45% (331 / 731) to 71% (518 / 731) (FIG. 44).

[0244] Overall, these analyses support the hypothesis that B-AR haplotype targeting is useful for increasing PgT indicated patient population, as well as bucindolol efficacy for the treatment of AF / HF and HF.* * *

[0245] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESReferences for Example 1:1. Bristow, M.R. (2011). 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Claims

1. WHAT IS CLAIMED IS:

1. A method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering an effective amount of bucindolol to a patient who has been genotyped and found to have at least one copy of a non-internalizing B-adrenergic receptor.

2. The method of claim 1, wherein the atrial fibrillation is new onset atrial fibrillation.

3. The method of claim 1 or 2, wherein the atrial fibrillation is recurrent atrial fibrillation.

4. The method of any one of claims 1 to 3, wherein the patient has, is suspected of having, or has been diagnosed with having heart failure.

5. The method of any one of claims 1 to 4, wherein at least one of the non-internalizing B-adrenergic receptors comprises a Bl -adrenergic receptor having a serine at amino acid position 49.

6. The method of any one of claims 1 to 5, wherein the patient has been found to have two copies of a Bl -adrenergic receptor having a serine at amino acid position 49.

7. The method of any one of claims 1 to 5, wherein at least one of the non-internalizing B-adrenergic receptors comprises a B2-adrenergic receptor not having a Gln27Argl6 haplotype.

8. The method of any one of claims 1 to 7, wherein the patient has been found to have one copy of a B2-adrenergic receptor that is not a Gln27Argl6 haplotype.

9. The method of any one of claims 1 to 7, wherein the patient has been found to have two copies of a B2-adrenergic receptor that is not a Gln27Argl6 haplotype.

10. The method of any one of claims 1 to 9, wherein the patient was further found to have at least one copy of a Bl -adrenergic receptor having an arginine at amino acid position 389.

11. The method of any one of claims 1 to 10, wherein the patient was further found to have at least one copy of a Bl -adrenergic receptor having a glycine at amino acid position 389.

12. The method of any one of claims 1 to 11, wherein the patient was further found to have at least two copies of a Bl -adrenergic receptor having an arginine at amino acid position 389.

13. The method of any one of claims 1 to 12, wherein the patient is administered about 12.5 to 200 mg of bucindolol per day.

14. The method of any one of claims 1 to 13, wherein the patient is administered about 0.15 to 5 mg / kg of bucindolol per day.

15. A method of treating, preventing, delaying onset of, or reducing the risk of cardiovascular mortality, the method comprising administering an effective amount of bucindolol to a patient who has been genotyped and found to have at least one copy of a noninternalizing B-adrenergic receptor.

16. A method of treating, preventing, delaying onset of, or reducing the risk of heart failure hospitalization, the method comprising administering an effective amount of bucindolol to a patient who has been genotyped and found to have at least one copy of a non-internalizing B- adrenergic receptor.

17. A method of treating, preventing, delaying onset of, or reducing the risk of all cause mortality, the method comprising administering an effective amount of bucindolol to a patient who has been genotyped and found to have at least one copy of a non-internalizing B-adrenergic receptor.

18. The method of any one of claims 15 to 17, wherein the patient has, is suspected of having, or has been diagnosed with having heart failure.

19. The method of any one of claims 15 to 18, wherein at least one of the non-internalizing B-adrenergic receptors comprises a Bl -adrenergic receptor having a serine at amino acid position 49.

20. The method of any one of claims 15 to 19, wherein the patient has been found to have two copies of a Bl -adrenergic receptor having a serine at amino acid position 49.

21. The method of any one of claims 15 to 20, wherein at least one of the non-internalizing B-adrenergic receptors is not a B2-adrenergic receptor having a Gln27Argl6 haplotype.

22. The method of any one of claims 15 to 21, wherein the patient was found to have at least two copies of a non-internalizing B-adrenergic receptor.

23. The method of any one of claims 15 to 22, wherein the patient was found to have at least two copies of a Bl -adrenergic receptor having a serine at amino acid position 49.

24. The method of any one of claims 15 to 23, wherein the patient was further found to have no copies of a B2-adrenergic receptor having a Gln27Argl6 haplotype.

25. The method of any one of claims 15 to 24, wherein the patient was further found to have at least one copy of a Bl -adrenergic receptor having an arginine at amino acid position 389.

26. The method of any one of claims 15 to 25, wherein the patient was further found to have at least two copies of a Bl -adrenergic receptor having an arginine at amino acid position 389.

27. The method of any one of claims 15 to 26, wherein the patient is administered about 12.5 to 200 mg of bucindolol per day.

28. The method of any one of claims 15 to 27, wherein the patient is administered about 0.15 to 5 mg / kg of bucindolol per day.

29. A method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering an effective amount of bucindolol to a patient identified as having at least one copy of an ADRB1 Arg389Ser49 haplotype.

30. The method of claim 29, wherein the patient is identified as having two copies of an ADRB1 Arg389Ser49 haplotype.

31. A method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering an effective amount of bucindolol to a patient identified as having at least one copy of an ADRB1 Gly389Ser49 haplotype.

32. The method of claim 31, wherein the patient is identified as having two copies of an ADRB1 Gly389Ser49 haplotype.

33. The method of any one of claims 29 to 32, wherein the patient is further identified as having two copies of an ADRB1 Ser49 genotype.

34. The method of any one of claims 29 to 33, wherein the patient is further identified as having no copies of an ADBR2 Gln27Argl6 haplotype.

35. The method of any one of claims 29 to 34, wherein the patient has, is suspected of having, or has been diagnosed with having heart failure.

36. The method of any one of claims 29 to 35, wherein the patient is administered about 12.5 to 200 mg of bucindolol per day.

37. The method of any one of claims 29 to 36, wherein the patient is administered about 0.15 to 5 mg / kg of bucindolol per day.

38. A method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering 12.5 to 200 mg of bucindolol to a patient who has been genotyped and found to have at least one copy of an ADRB1 Gly389Ser49 haplotype and no copies of an ADBR2 Gln27Argl6 haplotype.

39. A method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering 12.5 to 200 mg of bucindolol to a patient who has been genotyped and found to have at least one copy of an ADRB1 Arg389Ser49 haplotype and no copies an ADBR2 Gln27Argl6 haplotype.

40. A method of treating, preventing, delaying onset of, or reducing the risk of atrial fibrillation, the method comprising administering 12.5 to 200 mg of bucindolol to a patient who has been genotyped and found to have no copies of an ADBR2 Gln27Argl6 haplotype.

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