Treatment of acute heart failure

Abstract

The present invention provides ghrelin molecules, and corresponding methods and uses, for use in the treatment and / or prevention of acute heart failure (AHF) in an individual. The present invention further provides related compositions and kits of parts for use in the treatment and / or prevention of AHF in an individual.

Description

[Technical Field]

[0001] The present invention relates generally to the treatment of acute heart failure (AHF). [Background technology]

[0002] AHF is a clearly identifiable cardiac disease that involves a patient's rapid loss of cardiac function, which often leads to death. AHF is the most common cause of hospitalization (Ambrosy et al., 2014). Two particularly problematic types of AHF are acute decompensated heart failure (ADHF) and acute decompensated chronic heart failure (ADCHF).

[0003] In ADHF and ADCHF, hospital mortality rates range from 5 to 10%, with median hospital stays of 5 to 8 days. Over 50% of patients are discharged with unresolved symptoms, and within 30 or 60 days, studies have shown that half experience relapse, one-quarter are rehospitalized, and over 10% die (Baker et al., 2003; Curtis et al., 2008; Gheorghiade et al., 2006; Go et al., 2014; and Polanczyk et al., 2000). Population-wide mortality rates are 25 to 35% at 1 year (Lund, 2017). After improving outcomes in the late 1990s, prognosis for ADCHF has not improved since 2000 (Baker et al., 2003; Curtis et al., 2008; Polanczyk et al., 2000; and Thorvaldsen et al., 2016). The cost to society of heart failure is projected to triple between 2010 and 2030, with the majority of this cost being related to ADCHF (Heidenreich et al., 2013).

[0004] Currently, no treatments are available to reduce mortality or rehospitalization in patients with AHF, ADHF, or ADCHF. In some circumstances, diuretics are used to alleviate symptoms.

[0005] One particular challenge for treatment is that the condition makes patients highly sensitive to physiological changes such as arrhythmias, hypotension, and ischemia. This sensitivity means that some medications (such as inotropic agents) induce problematic physiological changes that lead to worse outcomes in AHF.

[0006] Therefore, AHF, especially ADHF and ADCHF, are difficult and expensive to treat, placing an economic burden on healthcare services. Therefore, there is a strong need to develop new and effective treatments for AHF, especially ADHF and ADCHF. Summary of the Invention

[0007] In this context, the present inventors have developed a new treatment for AHF, particularly ADHF and ADCHF, using ghrelin.

[0008] Thus, a first aspect of the present invention provides a ghrelin molecule for use in the treatment and / or prevention of acute heart failure (AHF) in an individual.

[0009] A second aspect of the present invention provides the use of a ghrelin molecule for the manufacture of a medicament for the treatment and / or prevention of acute heart failure (AHF) in an individual.

[0010] A third aspect of the present invention provides a method of treating and / or preventing acute heart failure (AHF) in an individual, the method comprising administering to the individual a ghrelin molecule.

[0011] AHF occurs in an individual when the function of the individual's heart (e.g., the heart's ability to maintain blood flow) deteriorates rapidly over a short period of time. An individual can be diagnosed with AHF if they exhibit rapid onset of symptoms associated with heart failure, such as shortness of breath (also called dyspnea), fatigue, cough, tissue swelling (also called edema), dizziness, and / or lightheadedness. If an individual with AHF does not receive treatment, the individual's heart will fail and the individual may die. A medically skilled artisan can identify whether an individual has AHF.

[0012] Ghrelin is a 28-amino acid peptide hormone. It is generally known to be secreted into the bloodstream from cells in the gastrointestinal tract of an individual and functions to regulate the distribution and rate of appetite and energy utilization. Ghrelin has been thought to act as an inotropic agent and exhibit potentially dangerous side effects in AHF patients, including cardiac arrhythmias, hypotension, and increased ischemia (Abraham et al., 2005; Cuffe et al., 2002; Mebasaa et al., 2007; and Packer et al., 2013). Because of these potentially dangerous side effects, ghrelin has never been considered suitable as a treatment for AHF.

[0013] In this context, the present inventors have now unexpectedly identified that ghrelin, while having an inotropic effect, does not exhibit the expected side effects and is suitable and highly effective for the treatment of AHF. The present inventors have found that ghrelin increases myocardial contractility, which is due to the Ca in cardiomyocytes. 2+ Instead of increasing the concentration of existing Ca 2+We have demonstrated the mechanism by which ghrelin functions: increasing the sensitivity of the heart to inotropic agents, thereby avoiding the previously predicted dangerous side effects (particularly cardiac arrhythmias, hypotension, and ischemia) associated with inotropic agents in patients with AHF. We also conducted a human study of ghrelin treatment in patients with less severe, advanced heart failure, a condition closely related to AHF, and found that ghrelin was effective in improving symptoms and cardiac output without worsening patient survival. For ethical reasons, we used patients with advanced heart failure instead of AHF in this study, as it would be safer to first test ghrelin in human patients with less severe heart failure symptoms. Detailed Description of the Disclosure

[0014] The terms "treatment" and "prophylaxis" are understood by those skilled in the medical arts.

[0015] "Treatment" includes any treatment of AHF in an individual, particularly a human, optionally including one or more of the following effects: (i) inhibiting AHF, e.g., delaying, reducing, or preventing the onset of AHF; (ii) alleviating AHF, e.g., causing regression of AHF in an individual suffering from AHF; (iii) curing AHF, e.g., restoring an individual suffering from AHF to a state of health in which no AHF is detectable; and / or (iv) reducing and / or inhibiting the progression and / or development of chronic heart failure (HF) when AHF is resolved.

[0016] "Prevention" includes any prophylactic treatment of AHF in an individual, particularly a human, optionally including one or more of the following effects: (i) preventing AHF from occurring in an individual (e.g., an individual who is predisposed to or at risk of developing AHF but has not yet been diagnosed with the condition), e.g., stopping an individual from developing AHF; (ii) delaying the onset of AHF in an individual, e.g., delaying an individual from developing AHF until later in the individual's life; (iii) limiting the occurrence of AHF in an individual, e.g., reducing the extent to which an individual is affected by AHF; (iv) preventing one or more symptoms of AHF in an individual, e.g., stopping an individual from developing one or more symptoms of AHF; and / or (v) Preventing complications of AHF, such as death of the individual and / or the occurrence of repeat episodes of AHF after the initial event has resolved and / or been cured.

[0017] In one embodiment, the individual is suspected of suffering from AHF.

[0018] Due to the rapid onset of AHF, in some circumstances it may be necessary to administer treatment such as with ghrelin before a diagnosis of AHF is confirmed, i.e., when AHF is only suspected. Thus, "suspected of having AHF" includes when an individual exhibits one or more of the signs and / or symptoms of AHF, but the individual has not yet been diagnosed with AHF.

[0019] In one embodiment, the individual is administered a therapeutically effective amount of a ghrelin molecule.

[0020] Those skilled in the art of medicine know what a "therapeutically effective amount" of ghrelin is, and a "therapeutically effective amount" includes an amount of ghrelin sufficient to effect beneficial and desired results, including clinical outcomes, associated with the treatment or prevention of AHF.

[0021] In one embodiment, the AHF is AHF on the left side of the heart, or alternatively AHF on the right side of the heart. Suitably, the AHF is AHF on the left side of the heart.

[0022] As those skilled in the medical arts will appreciate, the heart is anatomically divided into a left and right side. Each side of the heart is divided into two chambers: a ventricle and an atrium. Thus, the right side of the heart includes the right ventricle and right atrium, and the left side of the heart includes the left ventricle and left atrium. Deoxygenated blood enters the right atrium from the superior and inferior vena cava and then passes through the right ventricle. The blood then travels via the pulmonary circulation to the lungs, where it receives oxygen. Oxygenated blood then enters the left atrium, passes through the left ventricle, and from there is pumped into the systemic circulation via the aorta. Thus, "AHF of the right side of the heart" includes AHF in the right atrium and / or right ventricle. "AHF of the left side of the heart" includes AHF in the left atrium and / or left ventricle.

[0023] In one embodiment, the AHF is selected from the group including or consisting of acute decompensated heart failure (ADHF); hypertension-associated AHF; tachycardia-mediated AHF; pulmonary edema-associated AHF; cardiogenic shock AHF; or severe cardiogenic shock AHF.

[0024] Acute decompensated heart failure (ADHF) occurs in individuals who already suffer from heart failure or who are predisposed to heart failure, when the individual shows a rapid deterioration in cardiac function over a short period of time, which can be characterized by new heart failure symptoms and / or signs, worsening of existing heart failure symptoms and / or signs, and / or a sudden increase in the number of additional heart failure symptoms and / or signs. Particularly relevant symptoms and signs include shortness of breath, swelling of the legs, dizziness, unsteadiness, and / or fatigue. A medically skilled artisan can identify whether an individual suffers from ADHF.

[0025] AHF associated with hypertension is similar to the AHF described above, but its main symptom or sign is high blood pressure. A person skilled in the art of medicine can identify whether an individual suffers from AHF associated with hypertension.

[0026] Tachycardia-mediated AHF is similar to the AHF described above, but its primary symptom or sign is tachycardia. A person skilled in the art of medicine can identify whether an individual suffers from tachycardia-mediated AHF.

[0027] AHF associated with pulmonary edema is similar to the AHF described above, but its primary symptom or sign is pulmonary edema. A person skilled in the art of medicine can identify whether an individual suffers from AHF associated with pulmonary edema.

[0028] Cardiogenic shock AHF is similar to the AHF described above, but its main symptom or sign is cardiogenic shock. A person skilled in the art of medicine can identify whether an individual suffers from cardiogenic shock AHF.

[0029] Severe cardiogenic shock AHF is similar to the AHF described above, but its main symptom or sign is severe cardiogenic shock. A medically skilled artisan can identify whether an individual is suffering from severe cardiogenic shock AHF.

[0030] In a preferred embodiment, the acute decompensated heart failure (ADHF) is acute decompensated chronic heart failure (ADCHF) (also known as acute decompensated congestive heart failure) or congestive ADHF, more preferably acute decompensated chronic heart failure (ADCHF).

[0031] Acute decompensated chronic heart failure (ADCHF) occurs in individuals with pre-existing chronic heart failure (CHF). CHF is characterized by a gradual deterioration of cardiac function in an individual over an extended period of time (e.g., at least several months and often years), and can be diagnosed by the gradual worsening and / or appearance of symptoms and / or signs of heart failure. ADCHF is particularly relevant in individuals with CHF, which is characterized by symptoms such as dyspnea, edema, and / or fatigue. ADCHF occurs when an individual with CHF exhibits a rapid deterioration of cardiac function over a short period of time (e.g., over weeks to days). The prognosis for ADCHF is particularly poor because the patient is typically receiving treatment for CHF, but the condition worsens despite treatment. Those skilled in the art of medicine can identify whether an individual has ADCHF.

[0032] AHF (especially ADHF and ADCHF) differs from CHF because patients with CHF are stable at rest without dyspnea (shortness of breath) and have sufficient cardiac output to prevent deterioration of organ function. Conversely, patients with AHF (especially ADHF and ADCHF) have dyspnea even at rest and inadequate cardiac function, among other symptoms.

[0033] Other symptoms present in patients with AHF (especially ADHF and ADCHF) but not in patients with CHF include hypoxemia, worsening edema, rapid weight gain, elevated biomarkers (e.g., BNP and NT-proBNP) (e.g., elevated concentrations), worsening renal function (measured by worsening estimated glomerular filtration rate), hypotension, dizziness, and / or lightheadedness.

[0034] Congestive ADHF is similar to AHF and ADHF, and individuals with congestive ADHF may exhibit similar symptoms, although in congestive ADHF, individuals are particularly susceptible to fluid retention, which can cause shortness of breath, fatigue, and edema.

[0035] In another preferred embodiment, the acutely decompensated chronic heart failure (ADCHF) is severe ADCHF.

[0036] Severe ADCHF occurs in individuals with CHF whose cardiac function deteriorates very rapidly (e.g., over a period of days to hours). Severe ADCHF is characterized by individuals exhibiting hypotension, shock, arrhythmias, and widespread ischemia. Mortality rates among individuals with severe ADCHF are high, exceeding 50%. The prognosis for ADCHF is particularly poor because patients typically undergo treatment for CHF, which rapidly deteriorates despite treatment. Patients with severe ADCHF have a low chance of survival, even when treated with treatments known in the art (e.g., diuretics, oxygen, currently available inotropes and / or vasodilators). A medically skilled artisan can identify whether an individual has severe ADCHF.

[0037] In one embodiment, the individual has had CHF. A medically skilled artisan can determine whether an individual has CHF, for example, based on the individual's medical history. In some circumstances, the individual may have previously had or been diagnosed with CHF and may have fully recovered or been considered to have fully recovered. "Fully recovered" includes the individual being free of any CHF symptoms.

[0038] In one embodiment, the individual has suffered from progressive heart failure. Progressive heart failure is a progressive form of chronic heart failure, and is therefore sometimes referred to as progressive chronic heart failure. Progressive heart failure and progressive chronic heart failure are borderline stable forms of the disease. Unlike patients with AHF, patients with progressive heart failure or progressive chronic heart failure are usually treated at home rather than in a hospital. A medically skilled artisan can determine whether an individual has progressive chronic heart failure, for example, based on the individual's medical history. In some situations, the individual may have previously suffered from or been diagnosed with progressive chronic heart failure and may have recovered or been considered to have recovered. "Recovered" includes "fully recovered," in which the individual does not have any symptoms of progressive heart failure, as well as "partially recovered," in which the individual has a reduced number and / or severity of progressive heart failure symptoms. In other circumstances, an individual may have had or been diagnosed with progressive chronic heart failure immediately prior to the onset or diagnosis of AHF, including the individual having one or more symptoms of progressive heart failure immediately prior to the appearance of one or more AHF symptoms. In other words, progressive chronic heart failure worsens into AHF. It is understood that embodiments of the present invention may also be applied to the treatment of individuals currently suffering from progressive heart failure, even if the individual does not also suffer from AHF.

[0039] In one embodiment, the AHF is ADCHF or severe ADCHF, and the individual has been diagnosed with AHF for about 1 year or more, e.g., about 13 months or more; about 14 months or more; about 15 months or more; about 16 months or more; about 17 months or more; about 18 months or more; about 19 months or more; about 20 months or more; about 21 months or more; about 22 months or more; about 23 months or more; about 2 years or more; about 25 months or more; about 26 months or more; about 27 months or more; about 28 months or more; about 29 months or more; about 30 months or more; about 31 months or more; about 32 months or more; about 33 months or more; about 34 months or more; about 35 months or more; about 3 years or more; about 37 months or more; about 38 months or more; about 39 months or more; about 40 months or more; about 41 months or more months or more; about 42 months or more; about 43 months or more; about 44 months or more; about 45 months or more; about 46 months or more; about 47 months or more; about 4 years or more; about 49 months or more; about 50 months or more; about 51 months or more; about 52 months or more; about 53 months or more; about 54 months or more; about 55 months or more; about 56 months or more; about 57 months or more; about 58 months or more; about 59 months or more; about 5 years or more; about 66 months or more; about 6 years or more; about 78 months or more; about 7 years or more; about 90 months or more; about 8 years or more; about 102 months or more; about 9 years or more; about 114 months or more; or about 10 years or more, suitably about 2 years or more or about 3 years or more. In another embodiment, the AHF is ADCHF or severe ADCHF, and the individual has suffered from chronic heart failure (CHF) for a period of about 1 year or less; e.g., about 11 months or less; about 10 months or less; about 9 months or less; about 8 months or less; about 7 months or less; about 6 months or less; about 5 months or less; about 4 months or less; about 3 months or less; about 2 months or less; or about 1 month or less.

[0040] A medically skilled artisan can determine, for example, based on the individual's medical history, how long an individual had CHF before developing AHD or severe AHD. Thus, "AHD is AHD or severe AHD, and the individual had chronic heart failure (CHF)" includes the following: The individual has had CHF or one or more symptoms of CHF for a period of time prior to the onset of ADCHF or severe ADCHF; and / or The individual has been diagnosed with CHF for a period of time prior to the onset of ADCHF or severe ADCHF; and / or The individual has had CHF or one or more symptoms of CHF for a period of time prior to the diagnosis of ADCHF or severe ADCHF; and / or The individual had been diagnosed with CHF for a period of time prior to the diagnosis of ADCHF or severe ADCHF.

[0041] In one embodiment, the individual has previously suffered from AHF, suitably ADCHF. A medically skilled artisan can determine whether the individual has previously suffered from AHF, for example, based on the individual's medical history. In some circumstances, the individual may have previously suffered from or been diagnosed with AHF and may have recovered or been considered to have recovered. However, after the individual previously recovered from AHF, the individual is currently experiencing a recurrence of AHF.

[0042] Therefore, "an individual has previously suffered from AHF" includes: The individual previously fully recovered from AHF (e.g., the individual was previously free of any AHF symptoms and / or was no longer diagnosed with AHF), but the individual has now relapsed (e.g., the individual now has one or more AHF symptoms and / or has again been diagnosed with AHF); and / or The individual has previously partially recovered from AHF (e.g., the individual previously experienced a decrease in the number and / or severity of AHF symptoms), but is currently experiencing a relapse (e.g., the individual currently experiences an increase in the number and / or severity of AHF symptoms).

[0043] In one embodiment, the AHF is not associated with a myocardial infarction.

[0044] Myocardial infarction is also known as a heart attack. Myocardial infarction occurs when blood flow to a part of the heart is reduced or stopped, which damages the heart. A medically skilled artisan can distinguish whether an individual has suffered from myocardial infarction and whether AHF is associated with myocardial infarction. Thus, "AHF is not associated with myocardial infarction" includes AHF not caused by myocardial infarction; and / or the onset or diagnosis of AHF is not associated with myocardial infarction.

[0045] In a preferred embodiment, the individual has not been diagnosed as having suffered from a myocardial infarction and / or the individual is not suspected of having suffered from a myocardial infarction.

[0046] In one embodiment, the individual has not suffered a myocardial infarction for a period of about 1 month or less; for example, about 3 weeks or less; about 2 weeks or less; about 1 week or less; about 6 days or less; or about 5 days or less.

[0047] In one embodiment, the AHF is not nascent AHF, hi another embodiment, the AHF is nascent AHF.

[0048] De novo AHF is AHF in an individual with no prior history of heart failure, and occurs when the individual exhibits a rapid deterioration of cardiac function over a short period of time due to an acute cause (e.g., myocarditis). A medically skilled artisan can identify whether an individual suffers from de novo AHF.

[0049] In one embodiment, after administration of a ghrelin molecule, the individual experiences increased cardiac output; and / or increased cardiac contractility; and / or increased cardiac stroke volume; and / or improved ventricular function; and / or increased ventricular ejection fraction; and / or decreased phosphorylation of troponin I; and / or increased calcium sensitivity; and / or decreased intracellular cAMP; and / or improved renal function; and / or increased estimated glomerular filtration rate (eGFR); and / or improved dyspnea; and / or improved edema; and / or reduced biomarkers; and / or indicates one or more parameters from the group consisting of or including: reduction in hypotension; and / or resolution of cardiogenic shock; and / or reduction in dizziness; and / or reduction in lightheadedness; and / or reduction in arterial oxygen gradient (AVO2); and / or increase in pulmonary blood flow (PBF); and / or reduction in estimated systemic vascular resistance (eSVR); and / or reduction in pulmonary capillary wedge pressure; and / or reduction in left ventricular end-diastolic pressure; and / or reduction in left ventricular end-diastolic volume; and / or reduction in pulmonary artery pressure; and / or reduction in central venous pressure.

[0050] "Individual exhibits" includes a detectable and / or observable change in a particular parameter (such as cardiac output) in the individual, e.g., a detectable and / or observable increase in cardiac output in the individual following administration of a ghrelin molecule. A medically skilled artisan can identify whether an individual exhibits any of the aforementioned parameters.

[0051] A change in any parameter exhibited by an individual following administration of a ghrelin molecule is typically compared to a measurement of the same parameter prior to administration of the ghrelin molecule, e.g., cardiac output in an individual following administration of a ghrelin molecule is expected to be increased when compared to cardiac output in the individual prior to administration of the ghrelin molecule.

[0052] In a preferred embodiment, after administration of a ghrelin molecule, the individual exhibits one or more parameters from the group consisting of: increased cardiac output; and / or a decrease in arteriovenous oxygen (AVO2) gradient; and / or an increase in pulmonary blood flow (PBF); and / or a decrease in estimated systemic vascular resistance (eSVR). Because these particular parameters are particularly relevant to AHF, it will be particularly relevant if the individual exhibits any of these parameters as an indication that ghrelin is exerting its desired effect. In a more preferred embodiment, after administration of a ghrelin molecule, the individual exhibits an increase in cardiac output.

[0053] In one embodiment, the ghrelin molecule induces a change in one or more parameters in the individual, wherein the one or more parameters include an increase in cardiac output; and / or an increase in cardiac contractility; and / or an increase in cardiac stroke volume; and / or an improvement in ventricular function; and / or an increase in ventricular ejection fraction; and / or a decrease in troponin I phosphorylation; and / or an increase in calcium sensitivity; and / or a decrease in intracellular cAMP; and / or an improvement in renal function; and / or an increase in estimated glomerular filtration rate (eGFR); and / or an improvement in dyspnea; and / or an improvement in edema; and and / or reduction in biomarkers; and / or reduction in hypotension; and / or resolution of cardiogenic shock; and / or reduction in dizziness; and / or reduction in lightheadedness; and / or reduction in arterial oxygen gradient (AVO2); and / or increase in pulmonary blood flow (PBF); and / or reduction in estimated systemic vascular resistance (eSVR); and / or reduction in pulmonary capillary wedge pressure; and / or reduction in left ventricular end-diastolic pressure; and / or reduction in left ventricular end-diastolic volume; and / or reduction in pulmonary artery pressure; and / or reduction in central venous pressure.

[0054] "The ghrelin molecule causes a change" includes that a particular parameter is changed and / or altered in an individual after administration of the ghrelin molecule. A medically skilled artisan can identify whether a ghrelin molecule causes a change in any of the aforementioned parameters.

[0055] In a preferred embodiment, the ghrelin molecule causes a change in one or more parameters in the individual, wherein the one or more parameters are from the group comprising or consisting of: an increase in cardiac output; and / or a decrease in arteriovenous oxygen (AVO2) gradient; and / or an increase in pulmonary blood flow (PBF); and / or a decrease in estimated systemic vascular resistance (eSVR). In a preferred embodiment, the ghrelin molecule causes a change in the individual that is an increase in cardiac output.

[0056] Methods for measuring the parameters are known to those skilled in the medical arts.

[0057] In one embodiment, cardiac output is Cardiac output is about 1.5 L / min or more; for example, about 1.6 L / min or more; about 1.7 L / min or more; about 1.8 L / min or more; about 1.9 L / min or more; about 2 L / min or more; about 2.1 L / min or more; about 2.2 L / min or more; about 2.3 L / min or more; about 2.4 L / min or more; about 2.5 L / min or more; about 2.6 L / min or more; about 2.7 L / min or more; about 2.8 L / min or more; about 2.9 L / min or more; about 3 L / min or more; about 3.1 L / min or more; about 3.2 L / min or more; about 3.3 L / min or more; about 3.4 L / min or more; about 3.5 L / min or more; about 3.6 L / min or more; about 3.7 L / min or more; about 3.8 L / min or more; about 3.9 L / min or more; about 4 L / min or more; about 4.1 L / min or more; about 4.2 L / min or more; about 4.3 L / min or more; about 4.4 L / min or more; about 4.5 L / min or more; about 4.6 L / min or more; about 4.7 L / min or more; about 4.8 L / min or more; about 4.9 L / min or more; about 5 L / min or more; about 5.1 L / min or more; about 5.2 L / min or more; about 5.3 L / min or more; about 5.4 L / min or more; about 5.5 L / min or more; about 5.6 L / min or more; about 5.7 L / min or more; about 5.8 L / min or more; about 5.9 L / min or more; about 6 L / min or more; about 6.5 L / min or more; about 7 L / min or more; about 7.5 L / min or more; about 8 L / min or more; about 8.5 L / min or more; about 9 L / min or more; about 9.5 L / min or more; or about 10 L / min or more, preferably about 5.2 L / min or more; and / or Cardiac output is about 0.1 L / min or more; for example, about 0.15 L / min or more; about 0.2 L / min or more; about 0.25 L / min or more; about 0.3 L / min or more; about 0.35 L / min or more; about 0.4 L / min or more; about 0.45 L / min or more; about 0.5 L / min or more; about 0.55 L / min or more; about 0.6 L / min or more; about 0.65 L / min or more; about 0.7 L / min or more; about 0.75 L / min or more; about 0.8 L / min or more; about 0.85 L / min or more; about 0.9 L / min or more; about 0.95 L / min or more; about 1 L / min or more; about 1.05 L / min or more; about 1.1 L / min or more; about 1.15 L / min or more; about 1.2 L / min or more; about 1.25 L / min or more; about 1.3 or greater than about 1.35 L / min; or greater than about 1.4 L / min; or greater than about 1.45 L / min; or greater than about 1.5 L / min; or greater than about 1.55 L / min; or greater than about 1.6 L / min; or greater than about 1.65 L / min; or greater than about 1.7 L / min; or greater than about 1.75 L / min; or greater than about 1.8 L / min; or greater than about 1.85 L / min; or greater than about 1.9 L / min; or greater than about 1.95 L / min; or greater than about 2 L / min; or greater than about 2.5 L / min; or greater than about 3 L / min; or greater than about 4 L / min; or increased when the increase is about 5 L / min or greater, preferably about 0.5 L / min or greater, more preferably about 1.15 L / min or greater; and / or Cardiac output is about 5% or more, e.g., about 6% or more; about 7% or more; about 8% or more; about 9% or more; about 10% or more; about 11% or more; about 12% or more; about 13% or more; about 14% or more; about 15% or more; about 16% or more; about 17% or more; about 18% or more; about 19% or more; about 20% or more; about 25% or more; about 30% or more; about 35% or more; about 40% or more; about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; about 95% or more % or more; about 100% or more; about 105% or more; about 110% or more; about 115% or more; about 120% or more; about 125% or more; about 130% or more; about 135% or more; about 140% or more; about 145% or more; about 150% or more; about 155% or more; about 160% or more; about 165% or more; about 170% or more; about 175% or more; about 180% or more; about 185% or more; about 190% or more; about 195% or more; or about 200% or more, more preferably about 15% or more, and even more preferably about 25% or more.

[0058] One example of a method for measuring cardiac output is the use of inert gas rebreathing techniques, such as with the Innocor® device (Innovision, Odense, Denmark). Other techniques include echocardiography, Fick's technique, thermodilution, and indicator dilution, which are techniques known to those skilled in the art of medicine.

[0059] In one embodiment, cardiac output is measured using an inert gas rebreathing technique.

[0060] In another embodiment, cardiac output is measured by echocardiography, or Fick's method, or thermodilution, or indicator dilution.

[0061] In a preferred embodiment, cardiac output is increased when cardiac output is greater than or equal to about 5.2 L / min.

[0062] In another preferred embodiment, cardiac output is increased when cardiac output increases by about 0.5 L / min or more, more preferably by about 1.15 L / min or more.

[0063] In some embodiments, cardiac output can be measured using surrogate parameters, such as blood pressure, and / or urine output, and / or strong pulse, and / or weak pulse, and / or warm extremities (such as the arms and legs), and / or cold extremities (such as the arms and legs), and / or serum creatinine concentration, and / or estimated glomerular filtration rate. Those skilled in the art of medicine will know how measurements of these surrogate parameters can be used to demonstrate an increase in an individual's cardiac output.

[0064] In one embodiment, the cardiac contractility is and / or the increase in absolute fractional shortening is about 2% or more, e.g., about 3% or more; about 4% or more; about 5% or more; about 6% or more; about 7% or more; about 8% or more; about 9% or more; about 10% or more; about 11% or more; about 12% or more; about 13% or more; about 14% or more; about 15% or more; about 16% or more; about 17% or more; about 18% or more; about 19% or more; about 20% or more; about 25% or more; about 30% or more; about 35% or more; about 40% or more; about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; or about 95% or more, preferably about 3% or more; An increase occurs when the relative shortening fraction increases by about 20% or more, for example, about 25% or more; about 30% or more; about 35% or more; about 40% or more; about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; or about 95% or more, preferably about 42% or more.

[0065] The terms "absolute fractional shortening" and "relative fractional shortening" are known to those skilled in the art of medicine. An increase in absolute fractional shortening is the difference between the fractional shortening before and after administration (e.g., after administration and before administration of a ghrelin molecule) (e.g., fractional shortening after administration - fractional shortening before administration). An increase in relative fractional shortening is the difference between the fractional shortening before and after administration (e.g., after administration and before administration of a ghrelin molecule) divided by the previous value (e.g., (fractional shortening after administration - fractional shortening before administration) / fractional shortening before administration).

[0066] In a preferred embodiment, the cardiac contractility is increased when there is an increase in absolute fractional shortening of about 3% or more.

[0067] In another preferred embodiment, the cardiac contractility is increased when there is an increase in the relative shortening fraction of about 42% or greater.

[0068] In one embodiment, the cardiac stroke volume is If the cardiac stroke volume is greater than about 30 mL, e.g., greater than about 31 mL; greater than about 32 mL; greater than about 33 mL; greater than about 34 mL; greater than about 35 mL; greater than about 36 mL; greater than about 37 mL; greater than about 38 mL; greater than about 39 mL; greater than about 40 mL; greater than about 41 mL; greater than about 42 mL; greater than about 43 mL; greater than about 44 mL; greater than about 45 mL; greater than about 46 mL; greater than about 47 mL; greater than about 48 mL; greater than about 49 mL; greater than about 50 mL; greater than about 51 mL; greater than about 52 mL; greater than about 53 mL; greater than about 54 mL; greater than about 55 mL; greater than about 56 mL; greater than about 57 mL; greater than about 58 mL; greater than about 59 mL; greater than about 60 mL; greater than about 61 mL; greater than about 62 mL; greater than about 63 mL; greater than about 64 mL; greater than about 65 mL; greater than about 66 mL; greater than about 67 mL; greater than about 68 mL; greater than about 69 mL mL or more; about 70 mL or more; about 71 mL or more; about 72 mL or more; about 73 mL or more; about 74 mL or more; about 75 mL or more; about 76 mL or more; about 77 mL or more; about 78 mL or more; about 79 mL or more; about 80 mL or more; about 85 mL or more; about 90 mL or more; about 95 mL or more; or increased if about 100 mL or more, preferably about 69 mL or more, more preferably about 79 mL or more; and / or increased if cardiac stroke volume increases by more than about 3 mL, e.g., more than about 4 mL; more than about 5 mL; more than about 6 mL; more than about 7 mL; more than about 8 mL; more than about 9 mL; more than about 10 mL; more than about 11 mL; more than about 12 mL; more than about 13 mL; more than about 14 mL; more than about 15 mL; more than about 16 mL; more than about 17 mL; more than about 18 mL; more than about 19 mL; more than about 20 mL; more than about 25 mL; more than about 30 mL; more than about 35 mL; more than about 40 mL; more than about 45 mL; or more than about 50 mL; and / or An increase occurs when the cardiac stroke volume increases by about 5% or more, e.g., about 10% or more; about 15% or more; about 20% or more; about 25% or more; about 30% or more; about 35% or more; about 40% or more; about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; or about 95% or more, more preferably by about 15% or more, and even more preferably by about 25% or more.

[0069] In one embodiment, the cardiac stroke volume is increased when the stroke volume is between about 30 mL and about 100 mL, e.g., between about 40 mL and about 100 mL; between about 50 mL and about 100 mL; between about 60 mL and about 100 mL; between about 70 mL and about 100 mL; between about 80 mL and about 100 mL; or between about 90 mL and about 100 mL.

[0070] In a preferred embodiment, the cardiac stroke volume is increased when the stroke volume is greater than or equal to about 69 mL, more preferably greater than or equal to about 79 mL.

[0071] In another preferred embodiment, cardiac stroke volume is increased when cardiac stroke volume is increased by about 15% or more, more preferably by about 25% or more.

[0072] Cardiac stroke volume can be measured by echocardiography. In one embodiment, cardiac stroke volume is measured by echocardiography.

[0073] Cardiac stroke volume can be calculated by dividing cardiac output per minute by heart rate per minute. In one embodiment, cardiac stroke volume is calculated by dividing cardiac output per minute by heart rate per minute.

[0074] In one embodiment, the phosphorylation of troponin I is reduced by about 20% or more, e.g., about 25% or more; about 30% or more; about 35% or more; about 40% or more; about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; or about 95% or more, preferably by 50% or more.

[0075] In one embodiment, intracellular cAMP is reduced by about 20% or more, e.g., about 25% or more; about 30% or more; about 35% or more; about 40% or more; about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; or about 95% or more, preferably 75% or more.

[0076] As discussed herein, the inventors have identified that the mechanism by which ghrelin functions is through the reduction of intracellular cAMP, making it particularly beneficial when intracellular cAMP is reduced.

[0077] In one embodiment, the ventricular ejection fraction is and / or the absolute percentage increase is about 2% or more, e.g., about 5% or more; about 10% or more; about 15% or more; about 20% or more; about 25% or more; about 30% or more; about 35% or more; about 40% or more; about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; or about 95% or more, preferably about 5% or more or about 10% or more; and / or An increase in relative ratio occurs when the increase is about 15% or more, for example, about 16% or more; about 17% or more; about 18% or more; about 19% or more; about 20% or more; about 25% or more; about 30% or more; about 35% or more; about 40% or more; about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; or about 95% or more, preferably about 17% or more.

[0078] In preferred embodiments, the ventricular ejection fraction is increased when there is an absolute fraction increase of about 5% or more, about 10% or more.

[0079] In a preferred embodiment, the ventricular ejection fraction is increased when there is a relative fraction increase of about 25% or greater.

[0080] In one embodiment, the ventricular ejection fraction is increased when there is a relative fraction increase of about 20% to about 25%.

[0081] Ventricular ejection fraction can be measured by echocardiography. In one embodiment, ventricular ejection fraction is measured by echocardiography.

[0082] The terms "absolute rate" and "relative rate" are known to those skilled in the art of medicine. An absolute rate increase is the difference in percentage before and after (e.g., after administration of a ghrelin molecule and before administration) (e.g., post-administration percentage - pre-administration percentage). A relative rate increase is the difference before and after (e.g., after administration of a ghrelin molecule and before administration) divided by the previous value (e.g., (post-administration percentage - pre-administration percentage) / pre-administration percentage).

[0083] In one embodiment, the improvement in estimated glomerular filtration rate (eGFR) is an increase of about 10% or more, e.g., about 20% or more; about 30% or more; about 40% or more; about 50% or more; about 60% or more; about 70% or more; about 80% or more; or about 90% or more. Preferably, the improvement in estimated glomerular filtration rate (eGFR) is an increase of about 30% or more.

[0084] In one embodiment, the arteriovenous oxygen (AVO2) gradient is reduced by about 2% or more, e.g., about 3% or more; about 4% or more; about 5% or more; about 6% or more; about 7% or more; about 8% or more; about 9% or more; about 10% or more; about 15% or more; about 20% or more; about 25% or more; about 30% or more; about 35% or more; about 40% or more; about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; or about 95% or more, preferably by about 7% or more.

[0085] In one embodiment, the level of pulmonary blood flow (PBF) is about 0.1 L / min or greater, e.g., about 0.15 L / min or greater; about 0.2 L / min or greater; about 0.25 L / min or greater; about 0.3 L / min or greater; about 0.35 L / min or greater; about 0.4 L / min or greater; about 0.45 L / min or greater; about 0.5 L / min or greater; about 0.55 L / min or greater; about 0.6 L / min or greater; about 0.65 L / min or greater; about 0.7 L / min or greater; about 0.75 L / min or greater; about 0.8 L / min or greater; about 0.85 L / min or greater; about 0.9 L / min or greater; about 0.95 L / min or greater; about 1 L / min or greater; about 1.05 L / min or greater; about 1.1 L / min or greater; about 1.15 L / min or greater; about 1.2 L / min or greater; about 1.25 L / min or greater; about 1.3 L / min or more; about 1.35 L / min or more; about 1.4 L / min or more; about 1.45 L / min or more; about 1.5 L / min or more; about 1.55 L / min or more; about 1.6 L / min or more; about 1.65 L / min or more; about 1.7 L / min or more; about 1.75 L / min or more; about 1.8 L / min or more; about 1.85 L / min or more; about 1.9 L / min or more; about 1.95 L / min or more; or an increase of about 2 L / min or more, preferably about 0.8 L / min or more or about 1 L / min or more.

[0086] In one embodiment, the estimated systemic vascular resistance (eSVR) is about 100 dyn*s / cm -5 or more, for example, about 120 dyn*s / cm -5 Over 140 dyn*s / cm -5 Over 160 dyn*s / cm -5 Over 180 dyn*s / cm -5 Over 200 dyn*s / cm -5 Over 220 dyn*s / cm -5 Over 240 dyn*s / cm -5 Over 260 dyn*s / cm -5 Over 280 dyn*s / cm -5 Over 300 dyn*s / cm -5 Over 320 dyn*s / cm -5 Over 340 dyn*s / cm -5 Over 360 dyn*s / cm -5 Over 380 dyn*s / cm -5Over 400 dyn*s / cm -5 Over 420 dyn*s / cm -5 Over 440 dyn*s / cm -5 Over 460 dyn*s / cm -5 Over 480 dyn*s / cm -5 or approximately 500 dyn*s / cm -5 or more, preferably about 300 dyn*s / cm -5 It will decrease by more than this.

[0087] Systemic vascular resistance (eSVR) is a measurement known to those skilled in the art of medicine. It is a measure of the resistance that blood must overcome to reach the tissues. It can be calculated by dividing (arterial pressure - venous pressure) by cardiac output.

[0088] In one embodiment, AHF is caused by a spontaneous worsening of chronic heart failure; and / or an infection; and / or an allergic reaction; and / or a blood clot; and / or surgery; and / or cardiovascular disease; and / or pulmonary disease; and / or cardiomyopathy; and / or sleep apnea; and / or alcohol use; and / or illegal drug use; and / or anemia; and / or dyslipidemia; and / or hyperthyroidism; and / or Paget's disease; and / or hypertension (such as pulmonary hypertension); and / or prescription drug use; and / or smoking. and / or associated with one or more factors from the group including or consisting of: smoke; and / or hypertension; and / or renal dysfunction; and / or diabetes; and / or congenital heart disease; and / or lifestyle choices; and / or arrhythmia; and / or tachycardia; and / or bradycardia; and / or inflammation; and / or toxins; and / or autoimmune disease; and / or infiltrative disease; and / or connective tissue disease; and / or metabolic disease; and / or endocrine disease; and / or aging; and / or inherited genetic mutation; and / or pregnancy.

[0089] A medically skilled artisan can distinguish whether AHF is associated with the factors described above (e.g., infection). Thus, "associated with AHF" includes that AHF is caused by the factors described above; and / or the onset or diagnosis of AHF is associated with the factors described above.

[0090] In a preferred embodiment, AHF is associated with one or more factors from the group including or consisting of: spontaneous worsening of chronic heart failure; and / or infection; and / or diabetes; and / or lifestyle choices (such as an obese and / or sedentary lifestyle):

[0091] In another preferred embodiment, the AHF is associated with hypertension.

[0092] In one embodiment, the infection is a bacterial infection (such as bacterial pneumonia and / or bacterial sepsis), and / or a viral infection (such as viral pneumonia and / or viral sepsis), and / or a fungal infection, and / or a protozoan infection.

[0093] In one embodiment, the surgical procedure is from the group comprising or consisting of: cardiac surgery; and / or cardiac bypass surgery (such as retrograde arterial cardiac bypass surgery); and / or angioplasty; and / or heart valve repair surgery; and / or heart transplant surgery; and / or device implantation surgery (in some cases, the device is a pacemaker, and / or a heart pump, and / or a defibrillator).

[0094] In one embodiment, the cardiovascular disease is one or more of the group comprising or consisting of: coronary heart disease; and / or valvular disease; and / or coronary artery disease; and / or atherosclerosis; and / or stroke; and / or peripheral vascular disease.

[0095] In one embodiment, the pulmonary disease is asthma and / or chronic obstructive pulmonary disease (COPD).

[0096] In one embodiment, the prescribed medication is one or more of the group comprising or consisting of: diabetes medication; and / or calcium channel blockers; and / or nonsteroidal anti-inflammatory drugs (NSAIDs); and / or thiazolidinediones.

[0097] In one embodiment, the selected lifestyle is one or more of the group including or consisting of: poor diet; and / or lack of exercise; and / or being overweight; and / or obesity; and / or a sedentary lifestyle.

[0098] In one embodiment, the hyperthyroidism is associated with thyrotoxicosis.

[0099] In one embodiment, AHF is characterized by chest pain; and / or cough; and / or shock (such as cardiogenic shock); and / or hypertension; and / or oliguria; and / or anuria; and / or fatigue; and / or shortness of breath (dyspnea); and / or hypoxemia; and / or rapid breathing (tachypnea); and / or tachycardia; and / or ischemia; and / or edema (such as worsening edema); and / or renal dysfunction (such as worsening renal function); and / or hypotension; and / or organ failure (such as liver failure and / or renal failure); and / or cold extremities; and / or quadriplegia; and / or muscle fatigue; and / or nausea; and / or vomiting; and / or weight loss (such as anorexia); and / or pulmonary edema; and / or or one or more of the symptoms from the group including, or consisting of: lower body discomfort; and / or peripheral swelling; and / or hypoperfusion; and / or lower body swelling; and / or cardiac swelling; and / or weight gain (such as sudden weight gain); and / or weight loss; and / or cachexia; and / or bulging neck vessels (such as bulging neck veins and / or distended neck veins); and / or hepatomegaly; and / or dizziness; and / or syncope (also known as syncope); and / or altered mental status (e.g., anxiety and / or confusion and / or depression); and / or loss of appetite; hypotension; and / or cardiac arrhythmia; and / or difficulty sleeping; and / or discomfort when sitting up; and / or sleep apnea.

[0100] In preferred embodiments, AHF comprises one or more symptoms from the group including or consisting of: shortness of breath (dyspnea, e.g., dyspnea at rest); and / or hypoxemia; and / or edema (such as worsening edema); and / or sudden weight gain; and / or renal dysfunction (such as worsening renal function); and / or hypotension; and / or dizziness; and / or cardiogenic shock.

[0101] In another preferred embodiment, AHF comprises one or more of the symptoms from the group comprising or consisting of: shortness of breath (dyspnea); and / or discomfort when sitting; and / or rapid breathing; and / or anxiety; and / or hypoxemia; and / or hypertension.

[0102] In yet another preferred embodiment, the AHF comprises one or more of the following symptoms: edema (e.g., worsening edema); weight gain; and / or bulging neck veins; and / or hepatomegaly.

[0103] In one embodiment, the lower body comprises one or more of the group including or consisting of: the feet; and / or the ankles; and / or the legs (such as the lower and / or upper legs); and / or the hips.

[0104] In one embodiment, the sudden weight gain is and / or a weight gain of about 1 pound or more per day, e.g., about 2 pounds or more per day; about 3 pounds or more; about 4 pounds or more; about 5 pounds or more; about 6 pounds or more; about 7 pounds or more; about 8 pounds or more; about 9 pounds or more; or about 10 pounds or more, preferably 2 pounds or more per day or 3 pounds or more per day; and / or and / or about 1 pound or more per week, e.g., about 2 pounds or more per week; about 3 pounds or more; about 4 pounds or more; about 5 pounds or more; about 6 pounds or more; about 7 pounds or more; about 8 pounds or more; about 9 pounds or more; about 10 pounds or more; about 11 pounds or more; about 12 pounds or more; about 13 pounds or more; about 14 pounds or more; about 15 pounds or more; about 16 pounds or more; about 17 pounds or more; about 18 pounds or more; about 19 pounds or more; about 20 pounds or more; about 21 pounds or more; about 22 pounds or more; about 23 pounds or more; about 24 pounds or more; about 25 pounds or more; about 26 pounds or more; about 27 pounds or more; about 28 pounds or more; about 29 pounds or more; or about 30 pounds or more, preferably 5 pounds or more per week.

[0105] In a preferred embodiment, sudden weight gain comprises a weight gain of 2 pounds or more in one day.

[0106] In another preferred embodiment, sudden weight gain comprises a weight gain of 3 pounds or more in one day.

[0107] In yet another preferred embodiment, the sudden weight gain comprises a weight gain of 5 pounds or more in one week.

[0108] In one embodiment, the shortness of breath (dyspnea) is one or more of the group comprising or consisting of: shortness of breath at rest; and / or shortness of breath when walking; and / or shortness of breath when lying down; and / or exertional dyspnea; and / or orthopnea; and / or paroxysmal dyspnea; and / or nocturnal dyspnea.

[0109] In one embodiment, hypotension is a systolic blood pressure of about 40 mmHg or less, e.g., about 45 mmHg or less; about 50 mmHg or less; about 55 mmHg or less; about 60 mmHg or less; about 65 mmHg or less; about 70 mmHg or less; about 75 mmHg or less; about 80 mmHg or less; about 85 mmHg or less; about 90 mmHg or less; about 95 mmHg or less; about 100 mmHg or less; about 105 mmHg or less; about 110 mmHg or less; about 115 mmHg or less; or about 120 mmHg or less, preferably about 90 mmHg or less.

[0110] In one embodiment, low blood pressure is a systolic blood pressure of about 40 mmHg to about 120 mmHg.

[0111] In one embodiment, the cardiogenic shock is a blood pressure of about 45 mmHg or less, e.g., about 50 mmHg or less; about 55 mmHg or less; about 60 mmHg or less; about 65 mmHg or less; about 70 mmHg or less; about 75 mmHg or less; about 80 mmHg or less; about 85 mmHg or less; about 90 mmHg or less; about 95 mmHg or less; about 100 mmHg or less, preferably about 90 mmHg or less; and / or Approximately 3 L / min / m 2 Less than, for example, about 2.5 L / min / m 2 Less than; about 2 L / min / m 2Less than; approx. 1.5 L / min / m 2 or less than 1 L / min / m 2 or less, preferably about 2.5 L / min / m 2 cardiac index below; and / or a wedge pressure (e.g., pulmonary capillary wedge pressure) of about 5 mmHg or greater, e.g., about 10 mmHg or greater; about 15 mmHg or greater; about 20 mmHg or greater; about 25 mmHg or greater; or about 30 mmHg or greater, preferably about 15 mmHg or greater or about 16 mmHg or greater; and / or Cold extremities (e.g., cold toes and / or cold fingers); and / or oliguria; and / or Related to one or more of the group comprising or consisting of anuria.

[0112] In a preferred embodiment, cardiogenic shock is associated with a blood pressure of about 90 mmHg or less and a blood pressure of about 2.5 L / min / m 2 and / or wedge pressure of about 15 mmHg or greater; cold extremities; and / or oliguria; and / or anuria.

[0113] In one embodiment, ischemia occurs in one or more organs, for example in one or more organs of the group including or consisting of the heart; and / or brain; and / or kidney; and / or gastrointestinal tract; and / or liver, suitably in the heart.

[0114] Ischemia can occur due to inadequate blood flow, which can be caused by inadequate pumping of the heart, or by obstruction in the pulmonary system.

[0115] In one embodiment, the AHF is congestive ADHF, including symptoms of peripheral swelling and / or shortness of breath.

[0116] In one embodiment, the AHF is AHF associated with hypotension and includes one or more symptoms from the group including or consisting of: shortness of breath; and / or altered mental status; and / or oliguria; and / or anuria; and / or hypotension.

[0117] In one embodiment, the AHF is AHF associated with pulmonary edema and includes one or more symptoms from the group including or consisting of: shortness of breath; and / or rapid breathing; and / or tachycardia; and / or pulmonary edema.

[0118] In one embodiment, the AHF is AHF due to cardiogenic shock and includes one or more symptoms from the group including or consisting of: hypotension; and / or hypoperfusion; and / or oliguria; and / or cardiogenic shock; and / or altered mental status; and / or anuria.

[0119] In one embodiment, the AHF is AHF due to severe cardiogenic shock and comprises one or more symptoms from the group including or consisting of: hypotension; and / or hypoperfusion; and / or anuria; and / or oliguria; and / or severe cardiogenic shock; and / or altered mental status.

[0120] In one embodiment, the AHF is AHF of the right side of the heart, including symptoms of edema and / or bulging neck vessels.

[0121] In one embodiment, the AHF is severe ADCHF and includes one or more symptoms from the group including or consisting of: hypotension; and / or shock; and / or arrhythmia; and / or global ischemia.

[0122] In one embodiment, one or more symptoms develop over a period of about 1 month or less, e.g., about 3 weeks or less; about 2 weeks or less; about 7 days or less; about 6 days or less; about 5 days or less; about 4 days or less; about 3 days or less; about 2 days or less; or about 1 day or less, preferably about 7 days or less, more preferably from about 7 days to about 1 day.

[0123] "Development of one or more symptoms over a period of time" includes an increase in the number of symptoms and / or an increase in the severity of the symptoms over that period of time.

[0124] In one embodiment, the individual is diagnosed with AHF using one or more of the following procedures: X-ray examination; and / or blood tests; and / or electrocardiogram (ECG); and / or individual medical history tracing; and / or positron emission tomography (PET) scan; and / or multi-gated acquisition (MUGA) scan; and / or scintigraphy; and / or echocardiogram; and / or angiogram; and / or hemodynamic measurements; and / or computed tomography (CT) scan; and / or medical examination of symptoms; and / or biomarker (such as serum and / or plasma natriuretic peptide) measurements; and / or magnetic resonance imaging (MRI) scan.

[0125] Those skilled in the art of medicine know how to diagnose individuals with AHF using the procedures described above. For example, X-rays can be used to detect cardiac enlargement and fluid in the lungs or chest space. ECGs can be used to detect ischemia, tachycardia, bradycardia, and / or arrhythmias. PET scans, MUGA scans, scintigraphy, echocardiograms, angiograms, hemodynamic assessments, CT scans, and / or MRI scans can be used to detect (and / or measure and / or quantify) cardiac output, pulmonary blood flow, stroke volume, ejection fraction, cardiac contractility, and / or cardiac damage.

[0126] In a preferred embodiment, an individual is diagnosed with AHF using one or more of the following procedures: tracing the individual's medical history; and / or a medical examination of symptoms; and / or an echocardiogram; and / or serum natriuretic peptide measurements.

[0127] In one embodiment, the biomarker is one or more of the group comprising or consisting of serum natriuretic peptides; and / or sST2 cardiac biomarkers; and / or midregional proadrenomedullin (MR-proADM); and / or lactate, preferably serum natriuretic peptides. Preferably, the individual is diagnosed with AHF if one or more of the biomarkers serum and / or plasma natriuretic peptides; and / or sST2 cardiac biomarkers; and / or midregional proadrenomedullin (MR-proADM); and / or lactate are elevated.

[0128] A biomarker is "elevated" means that the biomarker in an individual is higher than would be expected in an individual without AHF. Those skilled in the art of medicine know how to detect these biomarkers and what concentrations of these biomarkers are considered elevated (e.g., based on Ponikowski 2016, ESC HF guidelines, European Heart Journal).

[0129] In one embodiment, the serum natriuretic peptides include B-type natriuretic peptide (BNP); and / or N-terminal pro-B-type natriuretic peptide (NT-proBNP); and / or midregional pro-atrial natriuretic peptide (MR-proANP).

[0130] In one embodiment, the individual: or about 170 ng / L or more; or about 180 ng / L or more; or about 190 ng / L or more; or about 200 ng / L or more, or about 250 ng / L or more, or about 300 ng / L or more, preferably about 100 ng / L or more; and / or A patient is diagnosed with AHF when NT-proBNP is about 250 ng / L or higher, for example, about 260 ng / L or higher; about 270 ng / L or higher; about 280 ng / L or higher; about 290 ng / L or higher; about 300 ng / L or higher; about 310 ng / L or higher; about 320 ng / L or higher; about 330 ng / L or higher; about 340 ng / L or higher; or about 350 ng / L or higher; or about 360 ng / L or higher; or about 370 ng / L or higher; or about 380 ng / L or higher; or about 390 ng / L or higher; or about 400 ng / L or higher; or about 425 ng / L or higher; or about 450 ng / L or higher; or about 500 ng / L or higher, preferably about 300 ng / L or higher.

[0131] In a preferred embodiment, an individual is diagnosed with AHF if BNP is greater than or equal to about 100 ng / L and / or NT-proBNP is greater than or equal to about 300 ng / L.

[0132] In one embodiment, an individual is diagnosed with AHF if MR-proANP is about 80 pmol / L or greater, e.g., about 90 pmol / L or greater; about 100 pmol / L or greater; about 110 pmol / L or greater; about 120 pmol / L or greater; about 130 pmol / L or greater; about 140 pmol / L or greater; or about 150 pmol / L or greater, preferably about 120 pmol / L or greater.

[0133] In one embodiment, the hemodynamic measurements are one or more of the group comprising: a measurement of systolic blood pressure; and / or a measurement of cardiac index; and / or a measurement of pulmonary capillary wedge pressure.

[0134] Pulmonary capillary wedge pressure can be measured by a pulmonary artery catheter. In one embodiment, pulmonary capillary wedge pressure is measured by a pulmonary artery catheter.

[0135] In one embodiment, the hemodynamic measurement is a measurement of systolic blood pressure, and the systolic blood pressure is greater than or equal to about 180 mmHg.

[0136] In one embodiment, the hemodynamic measurement is a measurement of systolic blood pressure, and the systolic blood pressure is less than or equal to about 90 mmHg.

[0137] In another embodiment, the hemodynamic measurement is a measurement of systolic blood pressure, and the systolic blood pressure is from about 90 mmHg to about 180 mmHg.

[0138] In one embodiment, the hemodynamic measurement is a measurement of cardiac index, and the cardiac index is about 5 L / min / m 2 Less than, for example, about 4.5 L / min / m 2 Less than; approx. 4 L / min / m 2 Less than; approx. 3.5 L / min / m 2 Less than; about 3 L / min / m 2 Less than; approx. 2.5 L / min / m 2 Less than; about 2 L / min / m 2 Less than; approx. 1.5 L / min / m 2 or less than 1 L / min / m 2 less than or equal to about 2 L / min / m 2 Less than or equal to approximately 1.8 L / min / m 2 The following is the result.

[0139] In one embodiment, the hemodynamic measurement is a measurement of pulmonary capillary wedge pressure, and the pulmonary capillary wedge pressure is about 10 mmHg or greater, e.g., about 11 mmHg or greater; about 12 mmHg or greater; about 13 mmHg or greater; about 14 mmHg or greater; about 15 mmHg or greater; about 16 mmHg or greater; about 17 mmHg or greater; about 18 mmHg or greater; about 19 mmHg or greater; about 20 mmHg or greater; about 25 mmHg or greater; about 30 mmHg or greater; about 35 mmHg or greater; or about 40 mmHg or greater, preferably about 15 mmHg or greater.

[0140] In one embodiment, after administration of a ghrelin molecule, the individual does not exhibit one or more of the following parameters: increased heart rate; and / or tachycardia; and / or decreased blood pressure; and / or hypotension; and / or increased oxygen demand; and / or ischemia; and / or increased plasma troponin T; and / or cardiac arrhythmia; and / or affected calcium transients. Those skilled in the art of medicine know how to measure these parameters. For example, heart rate can be measured using pulse palpation, an electrocardiogram (ECG), or telemetry. Blood pressure can be measured using an arterial cuff or line. As described herein, ischemia can be measured, inter alia, using BCG. Ischemia can be identified using clinical symptoms such as chest pain and / or chest pressure, as well as increased myocardial injury biomarkers (such as troponin T and / or troponin I).

[0141] "The individual does not exhibit" includes the absence of a detectable and / or observable change in a particular parameter (such as heart rate) in the individual, and / or this is clinically significant, e.g., the absence of a detectable or observable increase in the individual's heart rate following administration of a ghrelin molecule. A medically skilled artisan will be able to identify when an individual does not exhibit any of the aforementioned parameters.

[0142] Any change in a parameter that an individual does not exhibit after administration of a ghrelin molecule is typically compared to a measurement of the same parameter before administration of the ghrelin molecule, e.g., there is no increase in the individual's heart rate after administration of a ghrelin molecule when compared to the individual's heart rate before administration of the ghrelin molecule.

[0143] In a preferred embodiment, after administration of a ghrelin molecule, the individual does not exhibit one or more of the following: hypotension, and / or ischemia, and / or cardiac arrhythmia, and / or tachycardia. In a more preferred embodiment, after administration of a ghrelin molecule, the individual does not exhibit one or more of the following: hypotension, and / or ischemia, and / or cardiac arrhythmia. These particular parameters are particularly problematic side effects for patients with AHF, predicted to be caused by inotropy. Therefore, it would be particularly beneficial if ghrelin did not cause them.

[0144] In one embodiment, the ghrelin molecule does not cause a change in one or more parameters in an individual, said one or more parameters being from the group comprising or consisting of: increased heart rate; and / or tachycardia; and / or decreased blood pressure; and / or hypotension; and / or increased oxygen demand; and / or ischemia; and / or increased plasma troponin T; and / or cardiac arrhythmia; and / or affected calcium transients.

[0145] "The ghrelin molecule does not cause a change" includes that a particular parameter is not changed and / or altered in an individual after administration of the ghrelin molecule. A medically skilled artisan can distinguish whether a ghrelin molecule does not cause a change in any of the aforementioned parameters.

[0146] In a preferred embodiment, the ghrelin molecule does not cause a change in one or more parameters in an individual, said one or more parameters being from the group comprising or consisting of: hypotension, and / or ischemia, and / or cardiac arrhythmia, and / or tachycardia. In a more preferred embodiment, the ghrelin molecule does not cause a change in one or more parameters in an individual, said one or more parameters being from the group comprising or consisting of: hypotension, and / or ischemia, and / or cardiac arrhythmia.

[0147] Methods for measuring the parameters are known to those skilled in the medical arts.

[0148] In one embodiment, the heart rate is an increase of about 5 beats per minute or more, e.g., about 10 beats per minute or more; about 15 beats per minute or more; about 20 beats per minute or more; about 25 beats per minute or more; about 30 beats per minute or more; about 35 beats per minute or more; or about 40 beats per minute or more; and / or about 100 beats / minute or more, for example about 105 beats / minute or more; about 110 beats / minute or more; about 115 beats / minute or more; about 120 beats / minute or more; about 125 beats / minute or more; about 130 beats / minute or more; about 135 beats / minute or more; or about 140 beats / minute or more.

[0149] In one embodiment, the tachycardia is an increase in heart rate of about 10 beats / minute or more, e.g., 15 beats / minute or more; about 20 beats / minute or more; about 25 beats / minute or more; about 30 beats / minute or more; about 35 beats / minute or more; or about 40 beats / minute or more, preferably about 20 beats / minute or more; and / or A heart rate of about 100 beats / minute or more, for example about 110 beats / minute or more; 115 beats / minute or more; about 120 beats / minute or more; about 125 beats / minute or more; about 130 beats / minute or more; about 135 beats / minute or more; or about 140 beats / minute or more, preferably about 100 beats / minute or more.

[0150] In one embodiment, the tachycardia is sustained ventricular tachycardia and / or non-sustained ventricular tachycardia.

[0151] In one embodiment, blood pressure is reduced by about 5 mmHg or more, e.g., about 10 mmHg or more; about 15 mmHg or more; about 20 mmHg or more; about 25 mmHg or more; about 30 mmHg or more; about 35 mmHg or more; about 40 mmHg or more; about 45 mmHg or more; about 50 mmHg or more; about 55 mmHg or more; about 60 mmHg or more; about 65 mmHg or more; about 70 mmHg or more; about 75 mmHg or more; about 80 mmHg or more; about 85 mmHg or more; about 90 mmHg or more; about 95 mmHg or more; about 100 mmHg or more, preferably about 20 mmHg or more, more preferably between 20 mmHg and 80 mmHg. In a preferred embodiment, the reduction in blood pressure is independent of the individual's starting blood pressure.

[0152] In one embodiment, hypotension is a blood pressure of about 90 mmHg or less, e.g., about 85 mmHg or less; about 80 mmHg or less; about 75 mmHg or less; about 70 mmHg or less; about 65 mmHg or less; about 60 mmHg or less; about 55 mmHg or less; about 50 mmHg or less, preferably about 80 mmHg or less.

[0153] In one embodiment, the cardiac arrhythmia is selected from the group comprising or consisting of ventricular arrhythmia; supraventricular arrhythmia; ectopic atrial tachycardia; atrial flutter; sinus bradycardia; atrioventricular block (AV block); atrial fibrillation.

[0154] In one embodiment, ischemia comprises electrocardiogram (ECG) changes and / or increased plasma troponin T.

[0155] In one embodiment, the electrocardiogram (ECG) changes include one or more of the group comprising: ST elevation; and / or ST depression; and / or T wave changes. A person skilled in the art of medicine knows how to interpret these changes in the ECG and what they represent in the diagnosis and / or prognosis of an individual.

[0156] In one embodiment, plasma Troponin T increases by about 40% or more, e.g., about 45% or more; about 50% or more; about 55% or more; about 60% or more; about 65% or more; about 70% or more; about 75% or more; about 80% or more; about 85% or more; about 90% or more; or about 95% or more; or about 100% or more; or about 110% or more; or about 120% or more; or about 130% or more; or about 150% or more; or about 200% or more; or about 250% or more; or about 300% or more; or about 400% or more; or about 500% or more, preferably about 100% or more.

[0157] In one embodiment, the calcium transient comprises no change in amplitude of the calcium transient.

[0158] In one embodiment, the ghrelin molecule comprises one or more of the group including or consisting of: modified ghrelin; and / or a ghrelin fusion molecule; and / or a ghrelin fragment; and / or a ghrelin mutant; and / or a ghrelin derivative; and / or wild-type ghrelin.

[0159] Ghrelin (also known as lenomorelin (INN)) is a 28-amino acid peptide with a molecular weight of approximately 3,371 g / mol; in humans, it is encoded by the GHRL gene. Ghrelin is a peptide hormone produced by ghrelinergic cells in the gastrointestinal tract and functions as a neuropeptide in the central nervous system. Ghrelin binds to the ghrelin / growth hormone secretagogue receptor (GHS-R). The half-life of ghrelin in plasma is approximately 24–30 minutes. Normal human concentrations have been variously reported to range from 100–300 pmol / L (300–900 ng / L) for total ghrelin. Although data on acyl ghrelin are limited, we recently measured acyl ghrelin in 41 healthy, non-obese adults. The mean ± standard deviation of fasting acyl ghrelin was 118 ± 14 ng / L, with a range of 27–328 ng / L in the 5th–95th percentiles. 60 minutes after ingestion of a 260 kcal mixed meal, the lowest concentration was 80 ± 12 ng / L, with a range of 21–293 ng / L in the 5th–95th percentiles. Ghrelin is commercially available, for example, from Bachem (Bubendorf, Switzerland). The specific ghrelin product available from Bachem is a synthetic acylated human ghrelin under the trade name Clinalfa.

[0160] Ghrelin has the following amino acid sequence: Gly-Ser-Ser(octanoyl)-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys- Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-GIn-Pro-Arg (SEQ ID NO: 1).

[0161] In one embodiment, the ghrelin molecule specifically binds to and / or activates the ghrelin hormone secretagogue receptor (GHS-R).

[0162] "Modified ghrelin molecules" include chemically modified ghrelin molecules, such as derivatized ghrelin molecules. Chemical derivatives of one or more amino acids can be obtained by reaction with a functional side group. Such derivatized molecules include, for example, molecules in which free amino groups are derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carboxybenzoxy groups, f-butyloxycarbonyl groups, chloroacetyl groups, or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters, or other types of esters and hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. Chemical derivatives also include peptides containing naturally occurring amino acid derivatives of the 20 standard amino acids. For example, proline may be replaced with 4-hydroxyproline, lysine with 5-hydroxylysine, histidine with 3-methylhistidine, serine with homoserine, and lysine with ornithine. Derivatives may also include peptides containing one or more additions or deletions, so long as the requisite activity is maintained. Other modifications included are amidation, amino-terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g., with ammonia or methylamine), and similar terminal modifications.

[0163] For example, the modified ghrelin molecules described herein include not only molecules in which amino acid residues are linked by peptide (-CO-NH-) bonds, but also molecules in which the peptide bonds are reversed. Such retro-inverso peptidomimetics may be produced by methods known in the art, for example, as described in Meziere et al. (1997) J. Immunol. 159, 3230-3237, the disclosure of which is incorporated herein by reference. Such retro-inverso peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. Alternatively, the polypeptides of the present invention may be peptidomimetic compounds in which one or more amino acid residues are linked by -(OHNH)- bonds instead of the usual amide bonds.

[0164] It has been found that polypeptides can be conveniently hindered at their N- or C-termini, for example by amidation, to help reduce susceptibility to in vitro proteolytic digestion.

[0165] As described above, various unencoded or modified amino acids, such as D-amino acids and N-methyl amino acids, may be used to modify the ghrelin molecules of the present invention. Furthermore, predicted bioactive structures may be stabilized by covalent modifications, such as cyclization or incorporation of lactam or other types of bridges. Methods for the synthesis of cyclic homodetic and heterodetic peptides containing disulfide, sulfide, and alkylene bridges are disclosed in U.S. Patent No. 5,643,872. Other examples of cyclization methods are described and disclosed in U.S. Patent No. 6,008,058, the disclosures of which are incorporated herein by reference. A further approach to the synthesis of cyclic stabilized peptidomimetic compounds is ring-closing metathesis (RCM).

[0166] In summary, terminal modifications are well known to reduce susceptibility to proteinase digestion and are therefore useful for extending the half-life of peptides in solution, particularly in body fluids where proteases may be present. Polypeptide cyclization is also a useful modification, and is preferred because of the stable structure formed by cyclization and in view of the biological activity observed for cyclic peptides.

[0167] In one embodiment, the modified ghrelin is an acylated ghrelin molecule, which is also sometimes referred to as an activated ghrelin molecule or an octanoylated ghrelin molecule.

[0168] "Ghrelin fragments" include functional ghrelin fragments, such as those that specifically bind to and / or activate GHS-R and / or specifically bind to and / or activate other receptors that interact with ghrelin. In one embodiment, the fragments comprise about 5 or more amino acids of a wild-type ghrelin molecule, e.g., about 6 or more amino acids; about 7 or more amino acids; about 8 or more amino acids; about 9 or more amino acids; about 10 or more amino acids; about 11 or more amino acids; about 12 or more amino acids; about 13 or more amino acids; about 14 or more amino acids; about 15 or more amino acids; about 16 or more amino acids; about 17 or more amino acids; about 18 or more amino acids; about 19 or more amino acids; about 20 or more amino acids; about 21 or more amino acids; about 22 or more amino acids; about 23 or more amino acids; about 24 or more amino acids; about 25 or more amino acids; about 26 or more amino acids; or about 27 or more amino acids of a wild-type ghrelin molecule.

[0169] "Wild-type ghrelin" includes a ghrelin molecule having the amino acid sequence of a ghrelin molecule found endogenously in an organism, such as the 28 amino acid human ghrelin molecule. In one embodiment, the wild-type ghrelin molecule is one from the group comprising or consisting of: a rodent (e.g., mouse, and / or rat, and / or hamster, and / or guinea pig, and / or gerbil, and / or rabbit) wild-type ghrelin molecule; and / or a canine (e.g., dog) wild-type ghrelin molecule; and / or a feline (e.g., cat) wild-type ghrelin molecule; a primate (e.g., human, and / or monkey, and / or ape) wild-type ghrelin molecule; and / or an equine (e.g., horse) wild-type ghrelin molecule; and / or a bovine (e.g., cow) wild-type ghrelin molecule; and / or a porcine (e.g., pig) wild-type ghrelin molecule, preferably a human wild-type ghrelin molecule, more preferably a human wild-type ghrelin molecule having 28 amino acids.

[0170] "Ghrelin fusion molecules" include ghrelin molecules (such as modified ghrelin, and / or ghrelin fragments, and / or ghrelin mutants, and / or ghrelin derivatives, and / or wild-type ghrelin) fused to other polypeptides and / or proteins and / or peptides. For example, the ghrelin molecule may include one or more additional amino acids inserted internally and / or at the N-terminus and / or C-terminus.

[0171] "Ghrelin variants" include wild-type ghrelin molecules containing insertions, deletions, and / or substitutions, either conservative or non-conservative. In particular, variants of the polypeptide in which the alterations do not substantially alter the activity of the ghrelin molecule are included. In particular, ghrelin variants in which the alterations do not substantially alter the binding specificity to GHS-R and / or activation of GHS-R are also included. In one embodiment, the ghrelin variant has about 5% or more identity to the wild-type ghrelin molecule, e.g., about 10% or more identity to the wild-type ghrelin molecule; about 15% or more identity; about 20% or more identity; about 25% or more identity; about 30% or more identity; about 35% or more identity; about 40% or more identity; about 45% or more identity; about 50% or more identity; about 55% or more identity; about 60% or more identity; about 65% or more identity; or about 70% or more identity. identity; about 75% or more identity; about 80% or more identity; about 85% or more identity; about 86% or more identity; about 87% or more identity; about 88% or more identity; about 89% or more identity; about 90% or more identity; about 91% or more identity; about 92% or more identity; about 93% or more identity; about 94% or more identity; about 95% or more identity; about 96% or more identity; about 97% or more identity; about 98% or more identity; or about 99% identity. and / or comprises about 5 or more contiguous amino acids of a wild-type ghrelin molecule, e.g., about 6 or more contiguous amino acids; about 7 or more contiguous amino acids; about 8 or more contiguous amino acids; about 9 or more contiguous amino acids; about 10 or more contiguous amino acids; about 11 or more contiguous amino acids; about 12 or more contiguous amino acids; about 13 or more contiguous amino acids; about 14 or more contiguous amino acids; about 15 or more contiguous amino acids; about 16 or more contiguous amino acids; about 17 or more contiguous amino acids; about 18 or more contiguous amino acids; about 19 or more contiguous amino acids; about 20 or more contiguous amino acids; about 21 or more contiguous amino acids; about 22 or more contiguous amino acids; about 23 or more contiguous amino acids; about 24 or more contiguous amino acids; about 25 or more contiguous amino acids; about 26 or more contiguous amino acids; or about 27 contiguous amino acids.

[0172] The percentage of sequence identity between two polypeptides may be determined using a suitable computer program, such as the GAP program of the University of Wisconsin Genetic Computing Group, it being understood that the percentage of identity is calculated for polypeptides whose sequences are optimally aligned.

[0173] Alternatively, this sequence comparison may be performed using the Clustal W program (as described in Thompson et al., 1994, NucL Acid Res. 22:4673-4680, incorporated herein by reference).

[0174] Fragments and variants of the ghrelin molecule may be generated using any of the methods of protein engineering, directed evolution, and / or site-directed mutagenesis known in the art (see, e.g., Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press, the disclosure of which is incorporated herein by reference).

[0175] "Derivative ghrelin" includes potentially useful peptidomimetic compounds, such as peptidomimetics that specifically bind to and / or activate the GHS-R. The term "peptidomimetic" refers to compounds that mimic the structure and desirable characteristics of a particular ghrelin molecule as a therapeutic agent.

[0176] In one embodiment, the ghrelin molecule comprises one or more of the group comprising: a synthetic ghrelin molecule; and / or a recombinant ghrelin molecule; and / or an endogenous ghrelin molecule.

[0177] "Synthetic ghrelin molecules" include ghrelin molecules artificially produced without the use of host cells or host organisms, such as by liquid phase peptide synthesis and solid phase peptide synthesis.

[0178] In one embodiment, the synthetic ghrelin molecule is one or more from the group comprising: synthetic modified ghrelin; and / or synthetic ghrelin fusion molecule; and / or synthetic ghrelin fragment; and / or synthetic ghrelin mutant; and / or synthetic ghrelin derivative; and / or synthetic wild-type ghrelin.

[0179] A "recombinant ghrelin molecule" includes a ghrelin molecule produced by recombinantly expressing a gene encoding the ghrelin molecule in a recombinant host cell organism, such as a recombinant E. coli host, and preferably subsequently recovering and purifying the ghrelin molecule therefrom. In some embodiments, the recombinant ghrelin molecule does not include an N-terminal methionine amino acid.

[0180] In one embodiment, the recombinant ghrelin molecule is one or more from the group comprising or consisting of: recombinant modified ghrelin; and / or recombinant ghrelin fusion molecule; and / or recombinant ghrelin fragment; and / or recombinant ghrelin mutant; and / or recombinant ghrelin derivative; and / or recombinant wild-type ghrelin.

[0181] An "endogenous ghrelin molecule" includes a ghrelin molecule produced by a host organism, which may be a ghrelin molecule that is a wild-type ghrelin molecule for that host organism, or a ghrelin molecule that is not a wild-type ghrelin molecule for that host organism.

[0182] In one embodiment, the endogenous ghrelin molecule is one or more from the group including or consisting of endogenous modified ghrelin; and / or endogenous ghrelin fusion molecule; and / or endogenous ghrelin fragment; and / or endogenous ghrelin mutant; and / or endogenous ghrelin derivative; and / or endogenous wild-type ghrelin.

[0183] In one embodiment, the host organism is one from the group comprising or consisting of: rodents (e.g., mice, and / or rats, and / or hamsters, and / or guinea pigs, and / or gerbils, and / or rabbits); and / or canines (e.g., dogs); and / or felines (e.g., cats); primates (e.g., humans, and / or monkeys, and / or apes); and / or equines (e.g., horses); and / or bovines (e.g., cows); and / or porcines (e.g., pigs).

[0184] In one embodiment, the ghrelin molecule is administered one or more times per day, for example, two or more times per day; three or more times per day; four or more times per day; or five or more times per day, preferably twice per day.

[0185] In one embodiment, the ghrelin molecule is administered over a period of one day or more, for example, two days or more; three days or more; four days or more; five days or more; six days or more; one week or more; eight days or more; nine days or more; ten days or more; 11 days or more; 12 days or more; 13 days or more; two weeks or more; three weeks or more; four weeks or more; one month or more; five weeks or more; six weeks or more; seven weeks or more; eight weeks or more; two months or more; three months or more; four months or more; five months or more; six months or more; seven months or more; eight months or more; nine months or more; ten months or more; 11 months or more; or one year or more.

[0186] In one embodiment, the ghrelin molecule is administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every week, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 2 weeks, every 3 weeks, every 4 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or yearly.

[0187] In one embodiment, the ghrelin molecule is administered until a desired effect is achieved, such as: (i) inhibiting AHF, e.g., delaying, reducing, or preventing the onset of AHF; (ii) alleviating AHF, e.g., causing regression of AHF in an individual suffering from AHF; and / or (iii) curing AHF, e.g., restoring an individual suffering from AHF to a state of health in which AHF is no longer detectable.

[0188] In one embodiment, the ghrelin molecule is administered parenterally, and / or intravenously, and / or intraarterially, and / or intraperitoneally, and / or intrathecally, and / or intramuscularly, and / or subcutaneously, and / or by infusion.

[0189] In a preferred embodiment, the ghrelin molecule is administered by injection, such as intravenous infusion.

[0190] In one embodiment, the ghrelin molecule is administered by infusion over a period of about 10 minutes or more, e.g., about 20 minutes or more; about 30 minutes or more; about 40 minutes or more; about 50 minutes or more; about 1 hour or more; about 2 hours or more; about 3 hours or more; about 4 hours or more; about 5 hours or more; about 6 hours or more; about 7 hours or more; about 8 hours or more; about 9 hours or more; about 10 hours or more; about 11 hours or more; about 12 hours or more; about 13 hours or more; about 14 hours or more; about 15 hours or more; about 16 hours or more; about 17 hours or more; about 18 hours or more; about 19 hours or more; about 20 hours or more; about 21 hours or more; about 22 hours or more; about 23 hours or more; or about 24 hours or more, preferably about 6 hours or more or about 24 hours or more, more preferably 2 hours or more.

[0191] In one embodiment, the ghrelin molecule is administered at a rate of about 0.1 mL / min or more, e.g., about 0.2 mL / min or more; about 0.3 mL / min or more; about 0.4 mL / min or more; about 0.5 mL / min or more; about 0.6 mL / min or more; about 0.7 mL / min or more; about 0.8 mL / min or more; about 0.9 mL / min or more; about 1 mL / min or more; about 1.1 mL / min or more; about 1.2 mL / min or more; about 1.3 mL / min or more; about 1.4 mL / min or more; about 1.5 mL / min or more; about 1.6 mL / min or more; about 1.7 mL / min or more; about 1.8 mL / min or more; about 1.9 mL / min or more; about 2 mL / min or more; about 3 mL / min or more; about 4 mL / min or more; about 5 mL / min or more; about 6 mL / min or more; about 7 mL / min or more; about 8 mL / min or more; about 9 mL / min or more; or about 10 mL / min or more. It is administered by infusion at a rate of at least about 0.5 mL / min, preferably at a rate of at least about 0.5 mL / min.

[0192] In one embodiment, the ghrelin molecule is at least about 1 μg / kg body weight, e.g., at least about 2 μg / kg body weight; at least about 3 μg / kg body weight; at least about 4 μg / kg body weight; at least about 5 μg / kg body weight; at least about 6 μg / kg body weight; at least about 7 μg / kg body weight; at least about 8 μg / kg body weight; at least about 9 μg / kg body weight; at least about 10 μg / kg body weight; at least about 11 μg / kg body weight; at least about 12 μg / kg body weight; at least about 13 μg / kg body weight; at least about 14 μg / kg body weight; at least about 15 μg / kg body weight; at least about 16 μg / kg body weight; at least about 17 μg / kg body weight; at least about 18 μg / kg body weight; at least about 19 μg / kg body weight; at least about 20 μg / kg body weight; at least about 25 μg / kg body weight; at least about 30 μg / kg body weight; at least about 40 μg / kg body weight; at least about 50 μg / kg body weight; at least about 60 μg / kg body weight μg / kg body weight or more; about 70 μg / kg body weight or more; about 80 μg / kg body weight or more; about 90 μg / kg body weight or more; or about 100 μg / kg body weight or more, preferably about 12 μg / kg body weight or more.

[0193] In one embodiment, the ghrelin molecule is at about 0.001 μg / kg body weight / min or more, for example, about 0.002 μg / kg body weight / min or more; about 0.003 μg / kg body weight / min or more; about 0.004 μg / kg body weight / min or more; about 0.005 μg / kg body weight / min or more; about 0.006 μg / kg body weight / min or more; about 0.007 μg / kg body weight / min or more; about 0.008 μg / kg body weight / min or more; about 0.009 μg / kg body weight / min or more; about 0.01 μg / kg body weight / min or more; about 0.02 μg / kg body weight / min or more; about 0.03 μg / kg body weight / min or more; about 0.04 μg / kg body weight / min or more; about 0.05 μg / kg body weight / min or more; about 0.06 μg / kg body weight / min or more; about 0.07 0.08 µg / kg body weight / min or more; 0.09 µg / kg body weight / min or more; 0.1 µg / kg body weight / min or more; 0.2 µg / kg body weight / min or more; 0.3 µg / kg body weight / min or more; About 0.7 μg / kg or more; About 0.8 μg / kg or more; About 0.9 μg / kg or more; About 1 μg / kg or more; About 1.5 μg / kg or more; About 2 μg / kg or more; About 2.5 μg / kg or more; About 3 μg / kg or more; About 3.5 μg / kg or more More than μg / kg body weight / min; About 4.5 More than μg / kg body weight / minute; About 5 μg / kg body weight / min or more; about 5.5 μg / kg body weight / min or more; about 6 μg / kg body weight / min or more; about 6.5 μg / kg body weight / min or more; about 7 μg / kg body weight / min or more; about 7.5 μg / kg body weight / min or more; about 8 μg / kg body weight / min or more; about 8.5 μg / kg body weight / min or more; about 9 μg / kg body weight / min or more; about 9.5 μg / kg body weight / min or more; or about 10 μg / kg body weight / min or more, preferably about 0.1 μg / kg body weight / min or more.

[0194] In one embodiment, the ghrelin molecule is at least about 1 picomole (pmol), e.g., at least about 2 pmol; at least about 3 pmol; at least about 4 pmol; at least about 5 pmol; at least about 6 pmol; at least about 7 pmol; at least about 8 pmol; at least about 9 pmol; at least about 10 pmol; at least about 15 pmol; at least about 20 pmol; at least about 25 pmol; at least about 30 pmol; at least about 35 pmol; at least about 40 pmol; at least about 45 pmol; at least about 50 pmol; at least about 55 pmol; at least about 60 pmol; at least about 65 pmol; at least about 70 pmol; at least about 75 pmol; at least about 80 pmol; at least about 85 pmol; at least about 90 pmol; at least about 95 pmol; at least about 100 pmol; at least about 110 pmol; at least about 120 pmol; at least about 130 pmol; at least about 140 pmol; at least about 150 pmol or about 500 pmol or more, preferably about 30 pmol or more.

[0195] In one embodiment, the ghrelin molecule is administered at a concentration of about 1 μg / mL or more, such as about 1.2 μg / mL or more; about 1.4 μg / mL or more; about 1.6 μg / mL or more; about 1.8 μg / mL or more; about 2 μg / mL or more; about 2.2 μg / mL or more; about 2.4 μg / mL or more; about 2.6 μg / mL or more; about 2.8 μg / mL or more; about 3 μg / mL or more; about 3.2 μg / mL or more; about 3.4 μg / mL or more; about 3.6 μg / mL or more; about 3.8 μg / mL or more; about 4 μg / mL or more; about 4.2 μg / mL or more; about 4.4 μg / mL or more; about 4.6 μg / mL or more; about 4.8 μg / mL or more; about 5 μg / mL or more; about 5.2 μg / mL or more; about 5.4 μg / mL or more; about 5.6 μg / mL or more; about 5.8 μg / mL or more; about 6 μg / mL or more; about 6.2 μg / mL or more; about 6.4 μg / mL or more; about 6.6 μg / mL or more; about 6.8 μg / mL or more; about 7 μg / mL or more; about 7.2 μg / mL or more; about 7.4 μg / mL or more; about 7.6 μg / mL or more; about 7.8 μg / mL or more; about 8 μg / mL or more; about 8.1 μg / mL or more; about 8.2 μg / mL or more; about 8.3 μg / mL or more; about 8.4 μg / mL or more; about 8.5 μg / mL or more; about 8.6 μg / mL or more; about 8.7 μg / mL or more; about 8.8 μg / mL or more; about 8.9 μg / mL or more; about 9 μg / mL or more; about 9.1 μg / mL or more; about 9.2 μg / mL or more; about 9.3 μg / mL or more; about 9.4 μg / mL or more; about 9.5 μg / mL or more; about 9.6 μg / mL or more; about 9.7 μg / mL or more; about 9.8 μg / mL or more; about 9.9 μg / mL or more; about 10 μg / mL or more; about 11 μg / mL or more; about 12 μg / mL or more; about 13 μg / mL or more; about 14 μg / mL or more; about 15 μg / mL or more; about 16 μg / mL or more; about 17 μg / mL or more; about 18 μg / mL or more; about 19 μg / mL or more; about 20 μg / mL or more; about 21 μg / mL or more; about 22 μg / mL or more; about 23 μg / mL or more; about 24 μg / mL or more; about 25 μg / mL or more; about 26 μg / mL or more; about 27 μg / mL or more; about 28 μg / mL or more; about 29 μg / mL or more; or about 30 μg / mL or more.

[0196] In a preferred embodiment, the ghrelin molecule is administered at a concentration of about 10 μg / mL to about 30 μg / mL.

[0197] In one embodiment, the amount of ghrelin molecule administered is about 100 μg or more, e.g., about 120 μg or more; about 140 μg or more; about 160 μg or more; about 180 μg or more; about 200 μg or more; about 220 μg or more; about 240 μg or more; about 260 μg or more; about 280 μg or more; about 300 μg or more; about 320 μg or more; about 340 μg or more; about 360 μg or more; about 380 μg or more; about 400 μg or more; about 420 μg or more; about 440 μg or more; about 460 μg or more; about 480 μg or more; about 500 μg or more; about 520 μg or more; about 540 μg or more; about 560 μg or more; about 580 μg or more; about 600 μg or more; about 620 μg or more; about 640 μg or more; about 660 μg or more μg or more; about 680 μg or more; about 700 μg or more; about 720 μg or more; about 740 μg or more; about 760 μg or more; about 780 μg or more; or about 800 μg or more.

[0198] In a preferred embodiment, the ghrelin molecule is administered at a dose of about 0.1 μg / kg body weight / minute, at a concentration of about 10 μg / mL to about 30 μg / mL, and at an infusion rate of 0.5 mL / minute.

[0199] As outlined in Table 2 below, the concentration and / or amount of ghrelin administered may depend on the body weight of the individual to whom ghrelin is administered.

[0200] In therapy, the ghrelin molecules and compositions of the present invention may be administered alone, but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

[0201] The ghrelin molecules and compositions of the present invention are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solution should be suitably buffered (preferably to a pH of 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the medical arts.

[0202] Ghrelin molecules and compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0203] Ghrelin molecules and compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier, such as water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0204] A physician will determine the dosage of ghrelin molecules and compositions which will be most suitable for any individual, and the dosage will vary with the age, weight, and response of the particular individual.

[0205] In one embodiment, the individual is one from the group comprising or consisting of: rodents (e.g., mice, and / or rats, and / or hamsters, and / or guinea pigs, and / or gerbils, and / or rabbits); and / or canines (e.g., dogs); and / or felines (e.g., cats); primates (e.g., humans, and / or monkeys, and / or apes); and / or equines (e.g., horses); and / or bovines (e.g., cows); and / or porcines (e.g., pigs), preferably a human.

[0206] In one embodiment, the individual is 18 years of age or older, e.g., 20 years of age or older; 30 years of age or older; 40 years of age or older; 50 years of age or older; 60 years of age or older; 65 years of age or older; 70 years of age or older; 75 years of age or older; 80 years of age or older; 85 years of age or older; or 90 years of age or older, preferably 65 years of age or older.

[0207] AHF is generally considered to be a disease that is prevalent in elderly individuals, and therefore the ghrelin molecules of the present invention are likely to be particularly effective in elderly individuals.

[0208] In one embodiment, the ghrelin molecule is administered before, during, and / or after surgery.

[0209] In some situations, AHF may be caused in part by surgery, and therefore administration of the ghrelin molecules of the invention may be particularly effective before, after, or during surgery.

[0210] In one embodiment, the surgical procedure is selected from the group comprising or consisting of cardiac surgery; and / or cardiac bypass surgery (such as retrograde arterial cardiac bypass surgery); and / or angioplasty; and / or heart valve repair surgery; and / or heart transplant surgery; and / or gastrointestinal surgery; and / or orthopedic surgery; and / or neurosurgery; device implantation surgery (in some cases the device is a pacemaker, and / or a heart pump, and / or a defibrillator), and is suitably cardiac bypass surgery.

[0211] In one embodiment, the individual is administered one or more additional therapeutic agents.

[0212] In one embodiment, the ghrelin molecule is present in a composition, preferably a pharmaceutical composition.

[0213] In one embodiment, the composition comprises a pharmaceutically acceptable excipient.

[0214] In one embodiment, the composition comprises one or more members from the group comprising or consisting of: non-toxic organic acids; and / or non-toxic inorganic acids; and / or non-toxic organic bases; and / or non-toxic inorganic bases; and salts thereof.

[0215] Chemical bases that can be used as reagents to prepare pharmaceutically acceptable base salts of the present drugs, which are acidic in nature, are those that form non-toxic base salts with the compounds, including, but not limited to, salts derived from pharmaceutically acceptable cations, particularly alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts, such as N-methylglucamine meglumine, and lower alkanolammonium and other basic salts of pharmaceutically acceptable organic amines.

[0216] In one embodiment, the composition comprises one or more additional therapeutic agents.

[0217] In one embodiment, the one or more additional therapeutic agents are one or more additional therapeutic agents for the treatment of AHF.

[0218] In one embodiment, the one or more therapeutic agents are angiotensin-converting enzyme (ACE) inhibitors; and / or angiotensin II receptor blockers; and / or vasopressin receptor antagonists; and / or beta-blockers; and / or inotropic vasodilators (particularly milrinone and / or enoximone and / or dobutamine and / or levosimendan); and / or omecamtib mecarbil; and / or renin antagonists; and / or relaxin; and / or uralitide; and / or digoxin (lanoxin); and / or and / or vasodilators; and / or angiotensin II receptor antagonists (such as valsartan); and / or aspirin; and / or statins; and / or antihypertensives (such as sacubitril); and / or calcium sensitizers; and / or ivabradine; and / or diuretics; and / or vasopressors (such as noradrenaline, dopamine, vasopressin, and / or angiotensin II); and / or adenosine antagonists; and / or aldosterone antagonists.

[0219] In a preferred embodiment, the one or more therapeutic agents include valsartan and / or sacubitril.

[0220] In another preferred embodiment, the one or more therapeutic agents comprises a vasodilator.

[0221] In one embodiment, the calcium sensitizer comprises levosimendan and / or dobutamine.

[0222] In one embodiment, the vasodilator comprises a nitrovasodilator (such as nitroglycerin, and / or nitroprusside, and / or nesiritide).

[0223] In one embodiment, the diuretic comprises one or more of the group comprising or consisting of: furosemide; and / or bumetanide; and / or thiazides; and / or metolazone.

[0224] In one embodiment, the vasopressin receptor antagonist comprises tolvaptan and / or conivaptan.

[0225] In one embodiment, the adenosine antagonist comprises BG9719 and / or rolofylline.

[0226] In one embodiment, the ghrelin molecule is used in conjunction with a medical device, such as a heart pump. "Used in conjunction with" includes having one or more surfaces of the medical device coated with the ghrelin molecule.

[0227] A fourth aspect of the present invention provides a composition comprising a ghrelin molecule and one or more therapeutic agents selected from the list comprising: angiotensin-converting enzyme (ACE) inhibitors; and / or angiotensin II receptor blockers; and / or vasopressin receptor antagonists; and / or beta-blockers; and / or inotropic vasodilators (in particular milrinone and / or enoximone and / or dobutamine and / or levosimendan); and / or omecamtib mecarbil; and / or renin antagonists; and / or relaxin; and / or Uralitide; and / or digoxin (Lanoxin); and / or vasodilators; and / or angiotensin II receptor antagonists (such as valsartan); and / or aspirin; and / or statins; and / or antihypertensives (such as sacubitril); and / or calcium sensitizers; and / or ivabradine; and / or diuretics; and / or vasopressors (such as norepinephrine, dopamine, vasopressin, and / or angiotensin II); and / or adenosine antagonists; and / or aldosterone antagonists.

[0228] In a preferred embodiment (particularly of the fourth aspect), the one or more therapeutic agents are selected from the list comprising relaxin; and / or ularitide; and / or inotropic vasodilators; and / or vasopressors; and / or vasodilators.

[0229] In a more preferred embodiment, the one or more therapeutic agents include a vasodilator (particularly noradrenaline and / or vasopressin and / or angiotensin II). In another more preferred embodiment, the one or more therapeutic agents include an inotropic vasodilator (particularly milrinone and / or enoximone and / or dobutamine and / or levosimendan).

[0230] In one embodiment (particularly of the fourth aspect), the composition is used in conjunction with a medical device, such as a heart pump, where "used in conjunction with" includes having one or more surfaces of the medical device coated with ghrelin molecules.

[0231] In one embodiment of the fourth aspect of the invention, the composition comprises a pharmaceutically acceptable excipient.

[0232] The composition of the fourth aspect of the invention may comprise any one of the components described in the first, second and / or third aspects of the invention and may be used in any one of the uses or methods described in the first, second and / or third aspects of the invention.

[0233] A fifth aspect of the invention provides a kit of parts comprising a ghrelin molecule and / or composition according to the first, second, third and / or fourth aspects of the invention.

[0234] In an embodiment of the fifth aspect of the present invention, the kit of parts comprises instructions, which may be descriptive, instructional, marketing or other materials relating to the uses and / or methods and / or ghrelin molecules and / or compositions described herein.

[0235] The informational material in the kit is not limited in format. In one embodiment of the fifth aspect of the present invention, the informational material may include information regarding the production of the ghrelin molecule and / or composition, and / or the molecular weight of the ghrelin molecule and / or composition, and / or the concentration of the ghrelin molecule and / or composition, and / or the expiration date, and / or the batch, and / or the site of production. This information may be provided in a variety of formats, including printed documents, computer-readable materials, video or audio recordings, or information providing links or addresses to substantive materials, such as on the internet.

[0236] In a preferred embodiment of the fifth aspect of the invention, the kit of parts comprises instructions for the use of the ghrelin molecule and / or composition in the treatment of AHF.

[0237] In an embodiment of the fifth aspect of the invention, the kit comprises one or more containers for the ghrelin molecule and / or composition.

[0238] In an embodiment of the fifth aspect of the present invention, the kit of parts comprises one or more other items from the group comprising or consisting of: a solvent; and / or a buffer; and / or a stabilizer; and / or a preservative.

[0239] In an embodiment of the fifth aspect of the invention, the kit of parts comprises means for administering ghrelin molecules and / or compositions to an individual, preferably means for injecting ghrelin molecules and / or compositions into an individual.

[0240] The kit of parts of the fifth aspect of the invention may include any one of the arrangements described in the first, second, third and / or fourth aspects of the invention and may be for use in any one of the uses or methods described in the first, second, third and / or fourth aspects of the invention.

[0241] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an admission that the document is part of the state of the art or is common general knowledge. The present invention will now be described with reference to one or more non-limiting diagrams and examples. [Brief explanation of the drawings]

[0242] [Figure 1] Figure 1 is a harmonized flow diagram of patients screened and enrolled. [Figure 2] FIG. 2 is a flow diagram of the study and ghrelin / placebo infusions. [Figure 3] Figure 3 shows the acylated ghrelin concentration (ng / L). The notation "FU" indicates follow-up on days 2 to 5. [Figure 4] Figure 4 shows cardiac output (CO) before, during, and after ghrelin / placebo infusion. (A) In the ghrelin group, CO increased with infusion and decreased after cessation of infusion, with all pairwise comparisons being significant. In the placebo group, there was no significant change in CO. (B, C) The absolute and percent change in CO for individual patients differed between the ghrelin group (increase) and the placebo group (no change). [Figure 5] Figure 5 shows stroke volume (SV) before, during, and after ghrelin / placebo infusion. (A) In the ghrelin group, SV increased with infusion and decreased after cessation of infusion, with all pairwise comparisons being significant. In the placebo group, there was no significant change in SV. (B, C) Variation in absolute and percent change in SV within individual patients differed between the ghrelin group (increase) and the placebo group (no change). [Figure 6A] Figure 6A shows manually measured heart rate (HR) before, during, and after ghrelin / placebo infusion. (A) In the ghrelin group, HR decreased slightly from baseline at 120 minutes. In the placebo group, HR did not change significantly. (B, C) There were no statistically significant differences in absolute values ​​or percent changes in HR. [Figure 6B]Figure 6B shows the median heart rate from continuous monitoring with Nexfin. Since most patients were paced at 70 beats per minute with cardiac resynchronization therapy (biventricular pacemaker), the median heart rate is 70. For the mean values, see Figure 6C. Figure 6C shows the mean heart rate from continuous monitoring with Nexfin. [Figure 7] Figure 7 shows estimated systemic vascular resistance (SVR) before, during, and after ghrelin / placebo infusion. (A) In the ghrelin group, SVR decreased from baseline at 60 minutes. In the placebo group, SVR did not change significantly. (B, C) The absolute and percent change in SVR differed between the ghrelin group (decreased) and the placebo group (no change). [Figure 8] Figure 8 shows blood pressure measured by Nexfin. The systolic arterial pressure (Figure 8), diastolic arterial pressure (Figure 9), and mean arterial pressure (Figure 10) during and after ghrelin / placebo infusion are shown, as well as the median mean arterial pressure (Figure 11) and mean mean arterial pressure (Figure 12) measured continuously by Nexfin. There were no changes or differences between or within the ghrelin and placebo groups. [Figure 9] Figure 9 shows blood pressure measured by Nexfin. Systolic arterial pressure (Figure 8), diastolic arterial pressure (Figure 9), and mean arterial pressure (Figure 10) during and after ghrelin / placebo infusion are shown, as well as the median mean arterial pressure (Figure 11) and mean mean arterial pressure (Figure 12) continuously measured by Nexfin. There were no changes or differences between or within the ghrelin and placebo groups. [Figure 10] Figure 10 shows blood pressure measured by Nexfin. The systolic arterial pressure (Figure 8), diastolic arterial pressure (Figure 9), and mean arterial pressure (Figure 10) during and after ghrelin / placebo infusion are shown, as well as the median mean arterial pressure (Figure 11) and mean mean arterial pressure (Figure 12) measured continuously by Nexfin. There were no changes or differences between or within the ghrelin and placebo groups. [Figure 11]Figure 11 shows blood pressure measured by Nexfin. The systolic arterial pressure (Figure 8), diastolic arterial pressure (Figure 9), and mean arterial pressure (Figure 10) during and after ghrelin / placebo infusion are shown, as well as the median mean arterial pressure (Figure 11) and mean mean arterial pressure (Figure 12) measured continuously by Nexfin. There were no changes or differences between or within the ghrelin and placebo groups. [Figure 12] Figure 12 shows blood pressure measured by Nexfin. Systolic arterial pressure (Figure 8), diastolic arterial pressure (Figure 9), and mean arterial pressure (Figure 10) during and after ghrelin / placebo infusion are shown, as well as the median mean arterial pressure (Figure 11) and mean mean arterial pressure (Figure 12) measured continuously by Nexfin. There were no changes or differences between or within the ghrelin and placebo groups. [Figure 13] Figure 13 shows left ventricular end-diastolic diameter (LVEDD) from echocardiography. There were no changes or differences between or within the ghrelin and placebo groups. [Figure 14] Figure 14 shows left ventricular end-systolic diameter (LVESD). There were no changes or differences between or within the ghrelin and placebo groups. [Figure 15] Figure 15 shows left ventricular ejection fraction (LVEF). There was a trend toward greater absolute (p = 0.12) and percent change (p = 0.14) variation in EF with ghrelin, but no statistical changes or differences between or within the ghrelin and placebo groups. [Figure 16] Figure 16 shows tricuspid annular systolic excursion (TAPSE). The difference in change between ghrelin and placebo tended to be large. [Figure 17] Figure 17 shows E / e' (pulse wave Doppler diastolic mitral inflow velocity [E] / tissue Doppler diastolic mitral annular velocity [e], a surrogate for LV filling pressure). There was no difference in the change between ghrelin and placebo. [Figure 18]Figure 18 shows stroke volume (SV) measured by echocardiography. There was a significant difference in the change in SV for ghrelin (A). For ghrelin, there was a tendency for differences to appear in the absolute value and rate of change at 60 minutes, and a tendency for significant differences to appear in the absolute value and rate of change at 120 minutes. [Figure 19] Figure 19 shows cardiac output (CO) measured by echocardiography. There was a trend toward differences in the changes in CO with ghrelin (A), and there was a trend toward differences in the absolute values ​​and rate of change between 60 and 120 minutes with ghrelin (B). [Figure 20] Figure 20 shows the segmental strain measured by echocardiography. There was a difference in the values ​​for ghrelin, but it was not statistically significant. [Figure 21] FIG. 21 shows QTc during infusion and follow-up on days 2-5 relative to baseline (BL vs. FU). [Figure 22] Figure 22 shows HF hospitalization-free survival through 90 days. [Figure 23] FIG. 23 shows survival free of hospitalization for HF or the need for a heart transplant or left ventricular assist device through 90 days. [Figure 24] Figure 24 shows the contractility, as measured by fractional shortening (FS), of isolated cardiomyocytes from SHAM (control) and HF mice exposed to ghrelin, placebo, D-Lys (ghrelin antagonist), and D-Lys + ghrelin. Ghrelin enhances contractility in a ghrelin receptor-specific manner. Because this study was in vitro, these effects are independent of loading conditions (left ventricular preload or afterload). In this MI model, HF cardiomyocytes exhibit a compensatory response, i.e., a higher fractional shortening (FS). [Figure 25A]Figure 25A shows representative Ca2+ transients obtained from electrically stimulated adult cardiomyocytes. The amplitude of Ca2+ transients after ghrelin treatment of cardiomyocytes was not different between SHAM and HF mice. The left panel shows representative linear scan recordings of cardiomyocytes loaded with fluo-3, and the right panel shows representative cardiomyocyte Ca2+ transients. [Figure 25B] Figure 25B shows mean Ca transients from adult mouse cardiomyocytes. There was no difference between ghrelin and placebo in SHAM or HF mice. Data are presented as mean ± standard error. [Figure 26] Figure 26 shows the phosphorylation of cardiac troponin I. Immunoblots of protein lysates were performed using an antibody against the phosphorylation of serines 23-24 in troponin I. The left panel shows an example immunoblot of phosphorylated (serines 23-24) cardiac troponin I (cTnI phospho(23-24)) and total cardiac troponin I (cTnI). Treatment of cardiomyocytes with ghrelin was associated with decreased cTnI phosphorylation (hypophosphorylation) without altering total troponin I expression. Co-incubation with the ghrelin receptor antagonist D-Lys blocked the effect of ghrelin-induced hypophosphorylation. The right panel shows quantification of immunoblot band intensity, representing the phosphorylation level of cTnI in cardiomyocytes isolated from infarcted mouse hearts. Two animals were used, with one experiment per animal. Each experiment included four aliquots of cardiomyocytes isolated and treated as indicated. Mean values ​​± standard error of two experiments are shown. [Figure 27] Figure 27 shows the cAMP concentration in cardiomyocytes after incubation with ghrelin. Ghrelin reduces the cAMP concentration in cardiomyocytes. In the presence of the ghrelin receptor antagonist D-lys3, the effect of ghrelin on cAMP was blocked. [Example]

[0243] The following examples are included to demonstrate specific embodiments of the invention. Those of skill in the art will recognize that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to work well in the practice of the invention, and as such can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0244] Summary of test work Introduction Ghrelin is an endogenous appetite-stimulating peptide hormone with potential cardiovascular effects. Ghrelin has long been considered a potent inotropic agent, increasing intracellular Ca 2+ Based on this idea, it has been widely predicted that ghrelin would have many side effects that are highly dangerous for patients with acute heart failure (AHF), including increased cardiac arrhythmias, hypotension, and ischemia (Abraham et al., 2005; Cuffe et al., 2002; Mebasaa et al., 2007; and Packer et al., 2013).

[0245] Contrary to this interpretation, the present inventors unexpectedly found that ghrelin increases intracellular Ca 2+ It does not increase the concentration of existing Ca 2+ This led the inventors to believe that the previous assumption that ghrelin acts as a conventional inotropic agent and therefore causes hypotension, ischemia, and arrhythmias is incorrect, and that, contrary to previous misconceptions about ghrelin's function, ghrelin acts as a novel inotropic agent and can be used to treat AHF without causing hypotension, ischemia, or arrhythmias.

[0246] To test this hypothesis, we tested ghrelin in patients with heart failure. To avoid inadvertently harming patients tested with ghrelin, we conducted a clinical study in patients with advanced heart failure. While advanced heart failure is similar to AHF, it was anticipated that the severe adverse effects observed in AHF in response to inotropic agents would not occur to the same extent. Therefore, we believed that administering ghrelin to patients with advanced heart failure would provide indicators of safety and efficacy very similar to those observed in patients with AHF, but could be conducted in a safer manner.

[0247] As explained below, the present inventors have demonstrated that ghrelin acts as an inotropic agent, but that ghrelin also acts as a Ca 2+ Clinical studies have confirmed that ghrelin does not increase cardiac levels. Most importantly, the inventors demonstrated that ghrelin did not cause any of the side effects that are thought to be serious problems for patients with AHF, such as increased cardiac arrhythmias, hypotension, and ischemia. This experiment also showed that treatment with ghrelin led to improvement of symptoms that also occur in AHF, demonstrating that ghrelin is effective in treating the disease. Heart failure, its forms and outcomes

[0248] Heart failure (HF) is defined as insufficient cardiac output to meet metabolic demands or adequate CO2 secondary to compensatory neurohormonal activation.

[0249] Chronic HF (CHF) affects 2–3% of the population and up to 20% of the elderly ( Go et al., 2014 ) and is the most common cause of hospitalization (acute heart failure; AHF) ( Ambrosy et al., 2014 JACC 2014;63:1123-33).

[0250] AHF occurs most frequently when chronic compensation is inadequate and is called acute decompensated heart failure (ADHF). The majority of this occurs in patients with pre-existing CHF, hence the term acutely decompensated chronic heart failure (ADCHF).

[0251] Although medical and device therapies have improved outcomes in CHF with reduced ejection fraction (HFrEF), 5–10% of patients suffer from advanced (also called severe or end-stage) CHF, which is characterized by reduced cardiac output (CO), organ failure, frequent recurrent hospitalizations for ADCHF, and a high risk of death.

[0252] In ADHF and ADCHF, hospital mortality rates range from 5 to 10%, with median hospital stays of 5 to 8 days. Over 50% of patients are discharged with unresolved symptoms, and within 30 or 60 days, various studies have shown that half relapse, one-quarter are rehospitalized, and over 10% die (Baker et al., 2003; Curtis et al., 2008; Gheorghiade et al., 2006; Go et al., 2014; and Polanczyk et al., 2000). Population-wide mortality rates are documented as 25 to 35% at 1 year (Lund, 2017). After improving outcomes in the late 1990s, prognosis for CHF and ADCHF has not improved since 2000 (Baker et al., 2003; Curtis et al., 2008; Polanczyk et al., 2000; and Thorvaldsen et al., 2016). The cost to society of heart failure is projected to triple between 2010 and 2030, with the majority of this cost being related to ADCHF (Heidenreich et al., 2013).

[0253] Approximately 5% of hospitalizations for ADCHF have severe hemodynamic compromise with hypotension and shock, arrhythmias, and global ischemia (even in the absence of obstructive coronary artery disease), and short-term mortality in this group exceeds 50%. Inotropic therapy for advanced HF and ADCHF

[0254] Most inotropic agents increase intracellular Ca 2 + Some inotropes (e.g., milrinone, dobutamine) work by increasing the concentration of existing Ca 2+Although the former drug increases oxygen demand, and all drugs have multiple and complex mechanisms involving vasodilation, existing inotropic agents universally induce hypotension, tachyarrhythmias, and ischemia in clinical trials and practice, either with neutral effects on mortality or increased mortality (Abraham et al., 2005; Cuffe et al., 2002; Mebazaa et al., 2007; Packer et al., 2013). Three major classes of inotropic agents are used: phosphodiesterase (PD) inhibitors (e.g., milrinone); adrenergic agonists (e.g., dobutamine); and levosimendan. Specifically, in randomized, optime CHF trials, milrinone significantly increased hypotension and atrial arrhythmias and, although not significantly, increased mortality compared with placebo (Cuffe et al., 2002). In the observational ADHERE registry, both milrinone and dobutamine were independently associated with increased mortality (Abraham et al., 2005). Oral PD inhibitors, developed for long-term use, caused more arrhythmias, dizziness (likely due to hypotension), cardiac death, and sudden death compared with placebo (Amsallem et al., 2005; Cohn et al., 1998; Packer et al., 1991; Uretsky et al., 1990). Intermittent intravenous dobutamine infusion reduced hospitalizations for heart failure (Oliva et al., 1999), but increased mortality in a subsequent study that was stopped early and never published (Dies et al., 1986). In another study without a control group, only 3 of 13 patients survived the 26-week intermittent treatment study period (Krell et al., 1986). Levosimendan increases CO but also causes hypotension. It was neutral compared to dobutamine (which is harmful) in SURVIVE (Mebazaa et al., 2007), and increased hypotension and arrhythmias compared to placebo in REVIVE I and II (Packer et al., 2013).Thus, although existing inotropes worsen outcomes, they may be used in severe ADCHF and advanced CHF when organ function deteriorates and the patient cannot be mobilized for discharge, when cardiac transplantation is underway, and / or when death is imminent. Ghrelin

[0255] Ghrelin ("ghre" = growth) was first identified as a 28-amino acid peptide hormone that is an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHSR), acting in part by stimulating GH release (Kojima et al., 1999). Ghrelin has primarily been noted as a centrally acting appetite stimulant (Cummings et al., 2002). Ghrelin is released from the stomach in response to fasting and weight loss, but its release is inhibited by food ingestion (Kojima et al., 1999; Kojima and Kangawa, 2005; Shiiya et al., 2002). Ghrelin is acylated ("activated") at amino acid 3, and the acylated / acylated form is thought to be responsible for most of ghrelin's actions (Hosoda et al., 2000; Soares and Leite-Moreira, 2008).

[0256] Potential cardiovascular effects of ghrelin Ghrelin is elevated in cachectic CHF (Nagaya et al., 2001c) and non-cachectic CHF (Lund et al., 2009), but appears to be resistant to its appetite-stimulating effects, which resolve after heart transplantation (Lund et al., 2009). Beyond its metabolic effects, ghrelin appears to have specific cardiovascular actions. Ghrelin receptors (growth hormone secretagogue receptors; GHSRs) are widely distributed in cardiac and skeletal muscle and endothelium (Papotti et al., 2000). Ghrelin may be elevated in HF as a compensatory response to declining cardiac function, similar to the compensatory increase in catecholamines and natriuretic peptides. Indeed, ghrelin acylation is increased in HF, potentially representing an adaptive compensatory response, and decreases after heart transplantation (Zabarovskaja et al., 2014). Historical data on ghrelin treatment

[0257] In a rat model of HF, ghrelin increased CO and fractional shortening (Nagaya et al., 2001d); in a rat model of myocardial infarction, ghrelin reduced cardiac sympathetic nerve activity and left ventricular (LV) remodeling (Schwenke et al., 2008; Soeki et al., 2008) and apoptosis (Yang et al., 2014). Small studies in human HF have suggested that ghrelin can improve cardiac output (Nagaya et al., 2001b) and left ventricular ejection fraction (EF), exercise capacity, and muscle wasting (Nagaya et al., 2004), although these studies did not specify whether the form of ghrelin used was acylated or not. The cardiovascular effects of ghrelin have been reviewed, but data are inconsistent, showing both positive and negative inotropic effects (Soares et al., 2005), pointing to a variable and primarily nonacylated, inactive form of ghrelin (Broglio et al., 2003b; Isgaard, 2013; Kishimoto et al., 2012; Leite-Moreira et al., 2008; Nagaya and Kangawa, 2003a, b, 2006; and Nagaya et al., 2006). Furthermore, the safety, clinical efficacy, and mechanism of action of ghrelin in HF are unclear. GHSR agonists, such as pralmorelin and hexarelin, contain fewer amino acids and lack sequence similarity. Pralmorelin, but not ghrelin or hexarelin, improved CO in dogs with acute myocardial infarction (U.S. Patent No. 2004014671A1). Object of the invention

[0258] Based on unmet clinical need in acute HF (AHF, ADHF, ADCHF), we conducted a randomized, double-blind, placebo-controlled trial of intravenous acyl ghrelin in patients with heart failure. To determine the mechanisms underlying any clinical effects, we investigated the effects of ghrelin on contractility and cellular Ca in response to ghrelin in cardiomyocytes isolated from healthy control and HF mice. 2+ The transient response was evaluated.

[0259] Human clinical trial methods Study design and setup Between February 17, 2013, and May 19, 2015, a double-blind, placebo-controlled, parallel-group, single-center, prospective, randomized clinical trial was conducted with a single treatment with intravenous infusion of acyl ghrelin or placebo.

[0260] patient Patients were prospectively selected at the Heart Failure Clinic at Karolinska University Hospital (Figure 1) and had symptoms and signs of advanced CHF (New York Heart Association [NYHA] classification III-IV) and an EF <40%. Detailed inclusion and exclusion criteria are listed in Table 1.

[0261] [Table 1]

[0262] Preparation of ghrelin Ghrelin is a 28-amino acid peptide with a molecular weight of 3,371 g / mol. Its plasma half-life is 24-30 minutes. Normal human concentrations have been reported to vary from 100-300 pmol / L (300-900 ng / L) for total ghrelin. While data on acyl ghrelin are limited, we recently measured acyl ghrelin in 41 healthy, non-obese adults. Fasting levels were found to be a mean ± standard deviation of 118 ± 14 ng / L, with 5th-95th percentiles ranging from 27-328 ng / L. 60 minutes after ingestion of a 260 kcal mixed meal, the lowest concentration was 80 ± 12 ng / L, with 5th-95th percentiles ranging from 21-293 ng / L. Synthetic acylated (active) human ghrelin (trade name: Clinalfa, (human) ghrelin acetate, product number 4071265; Bachem, Hauptstrasse 144, 4416 Bubendorf, Switzerland; hereafter referred to as ghrelin) was purchased from Bachem (Bubendorf, Switzerland) under license from Daiichi Sankyo Co., Ltd. (Tokyo, Japan). For each patient and treatment, a stock solution consisting of multiple vials of powdered ghrelin (100 μg ghrelin / vial with phosphate buffer) was prepared. Each vial was dissolved in 1 mL of sterile water for injection (B. Braun, Germany) and visually inspected to ensure a clear, colorless solution. Then, 0.001 g (0.02 mL of 50 g / L stock solution) of human albumin (0.001 g / mL = 0.1% concentration) was added to each vial and further mixed with normal saline (9 mg / mL NaCl, B. Braun, Germany). The ratio of stock solution to saline was according to patient weight (Table 2), with a total volume of 100 mL (ensuring the same infusion volume for all patients). The final intravenous infusion rate was 0.50 mL / min (total volume 60 mL), corresponding to 0.1 μg (30 pmol) / kg / min of acyl ghrelin (total dose 12.0 μg / kg) administered to all patients randomized to ghrelin. The infusion was administered over 120 minutes and continued until all measurements were completed at 120 minutes (thus providing some additional ghrelin).The median total infusion time was 171 minutes. Measurements were repeated 30 minutes after the infusion was stopped.

[0263] [Table 2]

[0264] * indicates that the last data collection during the infusion was performed at 120 minutes. After all data had been collected, the infusion was stopped at a median of 171 minutes after the start of the infusion.

[0265] Placebo preparation Normal saline solution (9 mg / mL NaCl, B. Braun, Germany) was infused in the same volume, at the same rate (0.50 mL / min), and for the same total duration as the ghrelin infusion.

[0266] Procedures and data collection Potentially eligible patients who were pre-screened and provided written informed consent reported to the laboratory at 8:00 AM in a fasting state. The procedure is shown diagrammatically in Figure 2. Brachial artery blood pressure and oxygen saturation (peripheral pulse oximetry) were measured manually, and an EF of <40% was confirmed by echocardiography (visual estimation or Simpson's method). Two peripheral venous catheters were inserted, and blood samples were collected and analyzed, including estimated glomerular filtration rate (eGFR) and plasma glucose (as part of the inclusion / exclusion criteria). Patients who did not currently meet the eligibility criteria, including an EF of <40%, were excluded and considered misselected. Patients subsequently underwent additional testing and then consumed a 500 kcal standardized breakfast ad libitum without coffee or tea (because fasting increases endogenous ghrelin levels). Blood sampling and testing were repeated before the infusion began.

[0267] Patients were randomized in parallel to receive ghrelin or placebo via a four-group block randomization using paper envelopes, which were infused into the antecubital vein over 120 minutes. A dedicated study nurse prepared the ghrelin and placebo for infusion and blinded patients and all other study participants. Blood samples were collected immediately before, during, and after the 120-minute infusion, and 30 minutes after the infusion was stopped, to assess symptoms, signs, echocardiography, ECG, and noninvasive hemodynamics (Figure 2). Patients were discharged and returned for repeat measurements 2–5 days later. Patients were followed prospectively for morbidity and mortality outcomes.

[0268] Prespecified trial efficacy outcomes The primary efficacy outcome was the difference in CO change from the start of the infusion (time: 0 min) to the end of the infusion (time: 120 min) between ghrelin and placebo. A number of secondary outcomes related to hemodynamics, echocardiographic findings, and plasma biomarkers in response to the 120 min infusion, 30 min after infusion cessation, and 2–5 days after infusion cessation were also evaluated (see Results).

[0269] Pre-specified study safety outcomes Safety outcomes included decreases in systolic blood pressure, hypotension, and symptomatic hypotension during or after infusion, QTc prolongation, ischemia, and arrhythmias, and clinical outcomes.

[0270] Data collection methods and definitions Cardiac Output and Hemodynamics Noninvasive resting CO was assessed twice at each measurement using the Innocor® device (Innovision, Odense, Denmark). The Innocor is a noninvasive device that measures pulmonary blood flow using inert gas rebreathing and directly measures VO2. It has been validated for CO and VO2 at rest and during exercise (Gabrielsen et al., 2002; Stahlberg et al., 2009). It is similar to the gold standard Fick method (Warburton et al., 1999) and is superior to other noninvasive methods (Warburton et al., 1999), with a low coefficient of variation (VO2 < 2%; CO 5–7%) (Stahlberg et al., 2009). In the absence of significant intrapulmonary shunts, pulmonary blood flow calculated by inert gas rebreathing measured by Innocor® has been shown to provide a reliable estimate of CO (Stahlberg et al., 2009).

[0271] The Innocor® directly measures pulmonary blood flow, VO2, and SpO2. From these variables, shunt fraction, cardiac output, SVO2, and AV O2 difference were then calculated using standard formulas. Beat-to-beat ECG, blood pressure, and the first derivative of the pressure signal (+dP / dt) were measured using the Nexfin® device (described below). During Innocor measurements, 15 minutes of consecutive beat-to-beat Nexfin data were averaged. Estimated systemic vascular resistance (eSVR) was then calculated as mean blood pressure / cardiac output × 80. Stroke volume (mL) was calculated as cardiac output from Innocor / heart rate from Nexfin × 1000. Continuous hemodynamic and EKG monitoring

[0272] Blood pressure was measured using a plethysmograph-based (finger-cuff) technique (Nexfin®, BMEYE, Amsterdam, The Netherlands). Nexfin was also used to monitor heart rate. Heart rate and blood pressure data were measured continuously, beat-to-beat, throughout the entire study procedure. Data were then averaged over a 15-minute period after the Innocor measurement. In addition, EKGs were assessed before the start of the infusion, and 60 and 120 minutes after the infusion and after an ad libitum lunch. QT intervals were measured on the precordial lead V5 of a 12-lead surface ECG, with a paper feed speed of 50 mm / s and an amplitude of 10 mm / mV. Heart rate-corrected QT intervals (QTc) were calculated using the RR interval preceding the measured QT interval using the Bazett formula: QTc = QT / RR 1 / 2 The mean QTc from three heart beats was recorded.

[0273] Echocardiography Echocardiography was performed by a technician blinded to treatment assignment, and all echocardiograms were analyzed and interpreted by a single independent reviewer blinded to treatment assignment and patient history. Two-dimensional images were recorded using a Vivid 7 / E9 ultrasound system (GE, Horten, Norway) equipped with a 3-MHz Doppler transducer. A detailed echocardiogram, including dimensions, cardiac systolic and diastolic function, valvular performance, and systolic pulmonary artery pressure, was performed as a baseline (Table 5). Changes over time were compared using a simpler procedure, including left ventricular dimensions and function, stroke volume, and cardiac output (Tables 9 and 10). Left ventricular end-systolic and end-diastolic volumes and left ventricular ejection fraction (LVEF) were measured using a modified bidirectional Simpson method. LVEF was further measured using the Teichholz method. Because LVEF data from the Simpson method were missing due to poor image quality, changes in LVEF over time were calculated using the Teichholz method. Left atrial volume was calculated using the biplane area-length method from apical four-chamber and two-chamber views and indexed to body surface area. The E / e' ratio was calculated using the peak E-wave velocity of mitral inflow and the mean Doppler recordings of septal and lateral tissues. Tricuspid annular systolic excursion (TAPSE) was assessed by M-mode echocardiography. Speckle tracking was used for left ventricular longitudinal strain. Strain time course was calculated using the mean regional strain values ​​from the septal and inferior compartments. Stroke volume was derived from the left ventricular outflow tract (LVOT) area and the LVOT ventricular time integral (VTI) (LVOT area × LVOT VTI). Cardiac output was calculated as echo-derived stroke volume × heart rate.

[0274] [Table 3]

[0275] To summarize the abbreviations, LVEDD is left ventricular end-diastolic dimension; LVESD is left ventricular end-systolic dimension; LVESV is left ventricular end-systolic volume; LVEDV is left ventricular end-diastolic volume; LEVF is left ventricular ejection fraction; SV is stroke volume; CO is cardiac output; LV mass is left ventricular mass; LAV is left atrial volume; LAVi is left atrial volume index; RA is right atrium; MR is mitral regurgitation; TR is tricuspid regurgitation; AR is aortic regurgitation; MS is mitral stenosis; AS is aortic stenosis; TAPSE is tricuspid annular systolic excursion; SPAP is systolic pulmonary artery pressure; E is E-wave mitral inflow; E / e' is E-wave / e' ratio; and e' is mitral relaxation velocity.

[0276] [Table 4]

[0277] [Table 5]

[0278] Symptoms and Signs Headache, dizziness, dyspnea, central chest pain, facial flushing, drowsiness, upset stomach, and other symptoms were rated "yes" or "no" before the infusion, 30, 60, and 120 minutes after the infusion, 30 minutes after the infusion was completed, and at 2- to 5-day follow-up. Signs of rales, peripheral edema, jugular venous distension, hepatomegaly, and S3 gallop rhythm were recorded before the start of the infusion and after lunch.

[0279] blood sample Blood samples were collected 1) in the morning fasting state as a baseline, 2) after a standardized breakfast before treatment intervention, 3) 30 min after infusion, 4) 60 min after infusion, 5) 120 min after infusion, 6) 30 min after completion of infusion, and 7) at follow-up time points 2–5 days later (Figure 2). Blood was collected into ethylenediaminetetraacetic acid (EDTA) and serum tubes, immediately centrifuged, and plasma and serum aliquots were stored at -70°C until analysis.

[0280] For samples dedicated to ghrelin measurement, a protease inhibitor cocktail (50x stock solution) was prepared consisting of 5.5 p.i. and 10 mM KR-62436 (a DPP4 inhibitor) in DMSO and SIGMAFAST® protease inhibitor tablets (both from Sigma-Aldrich Corp., St. Louis, MO, USA) dissolved in distilled water at 2100 p.i. Blood samples were collected using 6 mL EDTA plasma tubes, immediately placed on ice, and 160 mL of the 50x protease inhibitor cocktail was added. The tubes were spun for 10 seconds and centrifuged at 2500 relative centrifugal force (RCF or g) for 10 minutes at 4°C. The resulting supernatant (plasma) was then pipetted into Eppendorf tubes (450 mL each) and immediately frozen at -70°C for storage until analysis.

[0281] Serum and plasma biomarkers Creatinine test values ​​were calculated according to the Cockcroft-Gault formula ([140 - age] x weight (kg) x 1.23) / creatinine x 0.85 (for women). Renal function was measured by creatinine and cystatin C in a central laboratory at Karolinska University Hospital. NT-proBNP was analyzed by proBNP II (Roche Diagnostics, Bromma, Sweden). Plasma glucose measurements were derived from EDTA-containing whole venous vasculature and analyzed by the photometric point-of-care technology, HemoCue® Glucose 201 RT (Angelholm, Sweden). Troponin T was analyzed in a central laboratory at Karolinska University Hospital.

[0282] Acyl ghrelin and pharmacokinetics Plasma concentrations of active (acylated) ghrelin were measured by a dedicated ELISA with electrochemiluminescence detection. Plasma samples were thawed and vortexed. They were then analyzed in duplicate on a 96-well multispot plate (Meso Scale Diagnostics, Rockville, MD, USA) coated with a capture antibody against acyl ghrelin according to the manufacturer's instructions. Plates were read using a Meso Scale Diagnostics Sector Imager 2400. The coefficient of variation (CV%) calculated from the curve-corrected plasma concentrations was 9.3 / 3.5 (intra- / inter-assay CV%). The lower limit of detection was 3.2 ng / L, and the upper limit was set at 10,000 ng / L.

[0283] Safety outcomes low blood pressure Hypotension was assessed in two ways: the difference in change in blood pressure between groups was quantified, and hypotension in any one patient was defined as a fall in systolic blood pressure of more than 20 mmHg or less than 80 mmHg.

[0284] arrhythmia Differences in heart rate changes between groups were quantified. Bradycardia was defined as a heart rate decrease of more than 15 beats / min to less than 40 beats / min, an increase in PQ interval of more than 20 milliseconds, or AV block II or III. In individual patients, tachycardia was defined as an increase in heart rate of more than 10 beats / min or a heart rate of more than 110 beats / min. Premature ventricular contractions were quantified, and ventricular tachycardia was defined as nonsustained if more than three beats in a row lasted for less than 30 seconds and sustained if it lasted for more than 30 seconds. Supraventricular arrhythmias were defined as ectopic atrial tachycardia, atrial flutter or atrial fibrillation, or AV nodal tachycardia lasting more than 3 seconds. Ghrelin may activate the hERG pathway, which could theoretically cause a prolongation of the QTc interval on EKG, predisposing to arrhythmias. Therefore, QTc was monitored.

[0285] ischemia Ischemia was assessed in two ways: the difference in troponin T change between groups was quantified, and ischemia in any one patient was defined as symptoms of ECG changes consistent with ischemia or an increase in troponin T of more than 50%.

[0286] Clinical outcomes Patients were followed within medical records until April 30, 2017. These outcomes excluded death from various causes, included all deaths or hospitalization for HF, all deaths or heart transplant or left ventricular assist device, and all combinations of these.

[0287] statistics For baseline characteristics, continuous variables were compared by Wilcoxon rank sum test for continuous variables and by Fisher's exact test for categorical variables.

[0288] To compare the effects of ghrelin versus placebo on the continuously variable overtime, a two-way repeated measures analysis of variance (2W-RM-ANOVA) was used to assess treatment effects within treatment groups. The 2W-RM-ANOVA had two factors: treatment level (two levels: ghrelin or placebo) and time level (three levels: baseline, 60 min post-infusion, and 120 min post-infusion). The primary analysis considered in this statistical model was the interaction (treatment × time), which tests the significance of the null hypothesis that the difference between treatments is the same at all time points. Because a time effect is expected, the time factor is largely irrelevant in this analysis. Because no differences between the ghrelin and placebo groups at baseline are expected, the treatment factor is largely irrelevant in this analysis. Post-hoc tests were performed to test for significant changes using Tukey's tests with adjusted p-values ​​for repeated measures within each treatment group and Sidak's tests between treatment groups. Differences between treatment groups at 60 and 120 minutes were also assessed using independent t-tests for absolute and %Δ values ​​at 60 and 120 minutes after the start of the infusion, within groups using the Wilcoxon signed-rank test and between groups using the Wilcoxon rank-sum test.

[0289] To compare time with outcome, survival and recurrence-free survival were plotted as Kaplan-Meier curves and compared by the log-rank test. Sample Size

[0290] Power calculations were based on the difference in CO change and were performed as described in Table 3. With 80% power, a two-sided alpha of 0.05, a sample size of 10 patients in each group was required to confirm the assumed minimal treatment difference of 10%. To account for potentially missing data measurements and additional margins, the sample size was set at 15 patients in each group.

[0291] [Table 6]

[0292] ethics Ghrelin has been previously studied in healthy humans and patients. This study was conducted in accordance with the International Conference on Harmonization and Good Clinical Practice guidelines and the Declaration of Helsinki. The study was approved by the local (Stockholm) ethics committee (approval numbers 2008 / 1:12 and 2008 / 1695-31). For this biomedical study, the ethics committee was exempt from the need for Medical Products Agency (MPA) approval based on a previous exemption for the same treatment in another study on gastrointestinal effects (MPA exemption number 159:2007 / 16373; Ethics Stockholm number 2007 / 119-31 / 1). All patients provided written informed consent.

[0293] Rodent study method A mouse model of myocardial infarction-induced heart failure Twelve-week-old C57BL6 mice were anesthetized with a gas mixture of oxygen and (2–3%) isoflurane. Myocardial infarction (MI) was induced in mice by permanent ligation of the left coronary artery, as previously described (Perrino et al., 2013). Briefly, mice underwent thoracotomy followed by left coronary artery ligation, and approximately 80% survived during the follow-up period (4–6 weeks). Sham (control) operated animals underwent the same procedure without coronary artery ligation. At the end of the study, mice were euthanized by cervical dislocation after administration of sedatives.

[0294] Transthoracic echocardiography in mice Cardiac function was monitored noninvasively in both groups by transthoracic echocardiography using a Philips HDI 5000 imaging system before termination. Echocardiography was performed using a 7-15 MHz CL15-7 scanhead. Cardiac contractility was measured as left ventricular fractional shortening (%) using the formula: LVd-LVs / LVd × 100, where LVd and LVs represent the diastolic and systolic dimensions of the left ventricle, respectively.

[0295] Isolation of mouse cardiomyocytes After euthanasia, single cardiomyocytes were isolated from the left and right ventricles (the mouse heart is innervated by the left ventricle) and the atria were excluded, following a procedure developed by the Alliance for Cellular Signaling (AfCS Procedure Protocol ID PP00000 125) as previously described (Pironti et al., 2016). Cytosolic [Ca] responses to ghrelin and placebo 2+ ] and cell shortening

[0296] Mouse cardiomyocytes were cultured with a cell-permeable form of the fluorescent indicator Fluo-3 AM, followed by a 5-minute washout period, as previously described (Andersson et al., 2011). Cardiomyocytes were seeded onto laminin-coated glass-bottom dishes. These dishes were placed in a custom-built perfusion / stimulation chamber and continuously perfused with Tyrode's solution aerated with O2 / CO2 (95 / 5%), containing the following composition: 121 mM NaCl, 5.0 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 0.4 mM NaH2PO4, 24 mM NaHCOs, 0.1 mM EDTA, and 5.5 mM glucose. Cardiomyocytes were stimulated to contract using an electric field between two platinum electrodes attached to the perfusion / stimulation chamber. Measurements were performed only in cardiomyocytes that contracted upon electrical stimulation and exhibited normal morphology (e.g., a striated "brick" shape). Cells exhibiting spontaneous contractions were not measured. Fluorescence was measured using a Bio-Rad MRC 1024 unit, a confocal microscope attached to a Nikon Diaphot inverted microscope (40-60x oil immersion lens). Confocal images were analyzed offline using ImageJ (National Institutes of Health; available at http: / / rsb.info.nih.gov / ij). Line-scan confocal images were obtained by line scanning along the long axis of paced cardiomyocytes. Free cytoplasmic Ca was measured. 2+ The change in emitted fluorescence, which represents the change in Ca, was quantified. 2+ The amplitude of the transient response was measured as the change in fluo-3 fluorescence signal (F) divided by the fluorescence immediately before the stimulation pulse applied under control conditions (F0). 2+ The transient decay time constant (τ) was quantified by fitting the decay to an exponential decay function in GraphPad Prism software (La Jolla, CA, USA). Cell shortening fraction was calculated from line-scan images as the rate of change in cell length at rest and at maximal contraction.

[0297] Myocytes were perfused with physiological buffer (Tyrode's solution) or, for pharmacological experiments, with Tyrode's plus ghrelin (100 nM) or D-Lys3 (a GHS-R1a antagonist, 3 μM). All cells were perfused with the respective solution for 15 min before measurements, except for some experiments in which D-Lys3 (3 μM) was introduced into the perfusion system 10 min before adding ghrelin. All experiments were performed at room temperature (approximately 24 °C).

[0298] Ghrelin treatment of cardiomyocytes Myocytes were perfused with physiological buffer (Tyrode's solution) with or without ghrelin (Bachem, Bubendorf, Switzerland) (100 nM). All cardiomyocytes were perfused with the respective solution for 15 min before measurements. An antagonist of the ghrelin receptor GHS-r1a (D-Lys3; 3 μM) was used in some experiments. In these experiments, D-Lys3 was introduced into the perfusion system 10 min before the addition of ghrelin.

[0299] Mouse cardiomyocyte protein immunoblot Isolated cardiomyocytes were treated with various conditions (placebo, ghrelin, D-Lys 3 GHS-R1a, or D-Lys 3 GHS-R1a + ghrelin) for 15 minutes. Cardiomyocytes were then pelleted and homogenized. Protein lysates were separated by electrophoresis and transferred to a membrane. The membrane was incubated with primary antibodies: rabbit phosphotroponin I (cardiac) (Ser 23 / 23) antibody (Cell Signaling #4004) and mouse troponin I (Millipore MAB1691A). Infrared-labeled secondary antibodies (IRDye 680 and IRDye 800, 1:5000, Licor) were then used. Immunoreactive zones were analyzed using an Odyssey infrared imaging system.

[0300] Band density was quantified with Image J and normalized to GAPDH, and final data were expressed as fold increase compared to the placebo group.

[0301] cAMP measurement Intracellular cAMP concentrations were measured using a cAMP direct immunoassay kit (Abeam ab65355). Briefly, frozen cardiomyocyte pellets from the above groups (placebo, ghrelin, D-Lys3 GHS-R1a + ghrelin) were homogenized on ice with a volume of 0.1 M HCl to obtain a protein concentration of 1 mg / mL. Acetylated supernatant samples were used for incubation with antibodies in ELISA plates according to the kit manufacturer's instructions. Finally, the optical density absorbance at 450 nm was analyzed using a microplate reader (Biotek, Synergy 2). The cAMP concentration was normalized by dividing the obtained reading (pmol / mL) by the total protein concentration (mg / mL) in each sample. Experiments were performed by an operator blinded to the animal treatments. statistics

[0302] Statistical comparisons between two groups were performed using the (unpaired) Student's t-test. Analysis of variance was used for comparisons between more than two groups. p<0.05 was used as the definition of statistical significance. Average data were expressed as the mean ± standard error of the mean (SEM).

[0303] ethical approval All animal experiments were carried out under ethical approval numbers N19 / 15, N273 / 15.

[0304] Results of human trials patient Thirty-four patients were identified from the Heart Failure Clinic at Karolinska University Hospital and consented to the study. On the morning of the study, one patient was excluded because of inability to rest due to neuropathy and leg pain, one patient was excluded because of a creatinine test value of 26 mL / min (less than 30 mL / min was an exclusion criterion), and one patient was excluded because of a New York Heart Association (NYHA) class II (NYHA class III-IV was the acceptance criterion) and an EF of 43% (an EF of less than 40% was the acceptance criterion). Thus, 31 patients were randomized. One patient (placebo group) experienced recurrent urinary urgency and dizziness early after the start of the infusion, leading to the interruption of the infusion and the patient's exclusion. Thirty patients remained in the analysis (15 in the ghrelin group; 15 in the placebo group).

[0305] Reference characteristics Baseline characteristics are shown in Table 4 and were similar between the treatment and placebo groups. Median age was 71 and 70 years, respectively, and 13% were women in both groups. Baseline echocardiographic data are shown in Table 5. Median EF (Simpson method) was 30% and 28%, respectively.

[0306] [Table 7-1] [Table 7-2] [Table 7-3]

[0307] To summarize the abbreviations, BMI is body mass index; SBP is systolic blood pressure; DBF is diastolic blood pressure; SpO2 is peripheral blood saturation by pulse oximetry; HF is heart failure; NYHA is New York Heart Association classification; INR is international normalized ratio; NT-proBNP is N-terminal pro-brain natriuretic peptide; hsCRP is high-sensitivity C-reactive protein; MI is myocardial infarction; AP is angina pectoris; CABG is coronary artery bypass graft; PCI is percutaneous coronary intervention; TIA is transient ischemic attack; CRT is cardiac resynchronization therapy; ICD is implantable cardioverter defibrillator; MRA is mineralocorticoid receptor antagonist; ACEi is angiotensin-converting enzyme inhibitor; ARB is angiotensin receptor blocker. *Based on file data from 41 healthy non-obese adults by D Webb.

[0308] [Table 8]

[0309] Patient outcomes Pharmacokinetics of plasma acyl ghrelin Plasma acyl ghrelin concentrations at baseline and during and after ghrelin infusion are shown in Table 6 and Figure 3. In the treatment group, median acyl ghrelin concentrations immediately reached near their maximum values. Acyl ghrelin levels were nearly normal 30 minutes after infusion and remained normal at follow-up 2–5 days.

[0310] [Table 9]

[0311] Primary efficacy outcome: cardiac output Tables 7 and 8 and Figure 4 show baseline CO and the responses to ghrelin and placebo. In the ghrelin group, CO increased with infusion and decreased after infusion was stopped, with all pairwise comparisons being significant. In the placebo group, CO did not change significantly, with p<0.0001 for the interaction. Absolute and percent changes in CO differed between the ghrelin and placebo groups. At 2- to 5-day follow-up, CO remained significantly increased with ghrelin compared with the placebo group (Table 8).

[0312] [Table 10]

[0313] [Table 11]

[0314] Secondary efficacy and safety outcomes: Hemodynamics Tables 7 and 8 and Figure 5 show the calculated stroke volume (SV) before, during, and after ghrelin / placebo infusion. In the ghrelin group, SV increased with infusion and decreased after cessation of infusion, with all pairwise comparisons being significant. In the placebo group, there were no significant changes in SV. The absolute change and percent change in SV differed between the ghrelin and placebo groups.

[0315] Tables 7 and 8 and Figure 6 show heart rates (HR) at selected time points, recorded manually and continuously by Nexfin. In the ghrelin group, HR decreased slightly between baseline and 120 minutes. In the placebo group, HR did not change significantly. There were no statistically significant differences in absolute or percent changes in HR.

[0316] Tables 7 and 8 and Figure 7 show the estimated systemic vascular resistance (SVR), and Figures 8-12 show the systolic, diastolic, and mean arterial blood pressures. The calculated SVR decreased proportionally with the increase in CO2 caused by ghrelin, but there was no change in blood pressure or the occurrence of hypotension.

[0317] Tables 7 and 8 show additional hemodynamic data. Oxygen consumption (VO2) was unchanged, suggesting that neither ghrelin nor placebo affected metabolism (oxygen demand). In the ghrelin group, pulmonary blood flow increased, consistent with the increase in CO.

[0318] Secondary outcome: echocardiography Tables 9 and 10 and Figures 13-20 show echocardiographic parameters before, during, and after infusion. There were no changes or differences in left ventricular end-diastolic diameter (LVEDD) (Figure 13) or left ventricular end-systolic diameter (LVESD) (Figure 14). For EF, which represents left ventricular function, there was a small interaction suggesting an improvement in EF with ghrelin, and a trend toward a difference in EF change between 60 and 120 minutes (Figure 15). For tricuspid annular systolic excursion (TAPSE), which represents right ventricular function, there was a trend toward an interaction suggesting an improvement in TAPSE, a trend toward a difference in change between 60 and 120 minutes, and a significant difference in absolute value at 120 minutes (Figure 16). There was no interaction or difference in change in E / e', a surrogate for LV filling pressure (Figure 17), suggesting that the increase in CO was not accompanied by a counter-inverse increase in filling pressure. For stroke volume (SV) measured by echocardiography, there was a significant interaction suggesting an improvement with ghrelin, which was consistent, albeit to a lesser extent, than for SV measured by noninvasive hemodynamics. There was also a difference in change in SV, but this was significant only at 120 minutes, not at 60 minutes (Figure 18). For cardiac output (CO) measured by echocardiography, there was a small interaction suggesting an improvement with ghrelin, but the change was not significant (Figure 19). The same results held for systolic strain, a surrogate for cardiac contractility (Figure 20).

[0319] Biomarkers Table 11 shows biomarkers before, during, and 30 minutes after infusion. There were no interactions with the biomarkers and no changes with troponin T. NT-proBNP increased in both groups, likely due to the prolonged supine position and lack of diuretic administration during the infusion. Cystatin C increased in both groups. Table 12 shows biomarkers on days 2 to 5 after infusion. NT-proBNP remained elevated in the ghrelin group. This is likely due to the stimulatory effect of ghrelin increasing its activity at or after the infusion. In multiple-infusion testing, this may also be due to chance.

[0320] Safety results are shown during and immediately after the infusion and for days 2–5 postinfusion. There was no effect of ghrelin or placebo on heart rate (Figure 6) or the incidence of ventricular tachycardia or bradyarrhythmia (not shown). There was no effect of ghrelin or placebo on systolic blood pressure (Figure 7), diastolic blood pressure (Figure 8), or mean arterial pressure (Figure 9). There were no events of hypotension or symptomatic hypotension in either group (not shown), except for one patient (placebo) who experienced repeated urinary urgency and dizziness early after the start of the placebo infusion, which resulted in the infusion being discontinued and this patient being excluded. There was no effect on troponin T (p-value by infusion interaction: 0.67 [Table 11] and p-value by day 2–5 follow-up interaction: 0.36 [Table 12]). There were no symptoms or EKG changes consistent with ischemia. One patient in the ghrelin group had a >50% increase in troponin T (baseline; 31, during infusion 34, 38, 42, 43, and 164 ng / L at follow-up, without symptoms of chest pain or ECG changes).

[0321] Among any symptoms rated "yes" or "no" (headache, dizziness, dyspnea, central chest pain, flushing, drowsiness, upset stomach, and other symptoms), flushing at 60 minutes was more frequent in the treatment group compared with placebo (7 vs. 0; p=0.006). QTc increased slightly, but not significantly (Table 13 and Figure 21).

[0322] [Table 12]

[0323] [Table 13]

[0324] Clinical safety outcomes at 90-day follow-up At 90-day follow-up, there were no differences in clinical events (Figures 22 and 23).

[0325] In vitro results of rodent cardiomyocyte studies Inotropic effects of ghrelin on healthy and failing cardiac myocytes in mice After 15 min of ghrelin exposure, the fractional shortening (FS) of cardiomyocytes was increased compared to placebo. The ghrelin-induced increase in contractility was observed in cardiomyocytes from both control and post-MI HF mice (Figure 24). The effect of ghrelin on FS was blocked by pretreatment with a GHS receptor 1a antagonist (D-Lys 3; Sigma). This suggests that the inotropic effect of ghrelin in cardiomyocytes is mediated by GHS-R1a.

[0326] The inotropic effect of ghrelin is due to Ca 2+ Due to sensitization of Cardiomyocyte Ca 2 + The transient responses did not differ among the various treatments (placebo, ghrelin, D-Lys3, or D-Lys3 + ghrelin) (Figures 25A and 25B). 2 + The rate of increase in the transient response and τ (decay time constant) were similar between groups, suggesting that ghrelin treatment significantly increased Ca 2 + The kinetics of the transient response was not affected by ghrelin (Fig. 25A). Collectively, these data suggest that the contractile-enhancing effect of ghrelin is mediated by intracellular Ca 2+ is mediated by an increase in myofibrillar Ca 2 + These findings suggest that this may be mediated by increased susceptibility to

[0327] Ghrelin-induced Ca 2+ Sensitization to troponin I is associated with decreased phosphorylation of troponin I Post-translational modifications of myofibrillar proteins, such as troponin, increase myofibrillar Ca 2 + For example, phosphorylation of serine 23-24 of troponin I regulates the sensitivity of myofibrillar Ca 2 + This is associated with decreased sensitivity to troponin I (Hasenfuss and Teerlink, 2011; Layland et al., 2005). To test whether ghrelin alters protein phosphorylation, we used an antibody targeting the phosphorylated serine 23-24 residues of troponin I. Protein lysates from healthy cardiomyocytes treated with placebo, ghrelin, or D-Lys3+ ghrelin were used. In the presence of ghrelin, the phosphorylation signal was lower compared to placebo (Figure 26). Pretreatment of cardiomyocytes with the ghrelin receptor antagonist D-Lys3 prevented the decrease in serine 23-24 phosphorylation in troponin I (Figure 26). This suggests that the inotropic effect of ghrelin may involve post-translational modifications of troponin.

[0328] Ghrelin decreases cAMP levels and troponin I phosphorylation in cardiac myocytes cAMP is a key molecule for intracellular signaling between plasma membrane receptors (e.g., β-adrenergic receptors) and for regulating muscle cell contractility. cAMP activates protein kinase A (PKA), which can in turn phosphorylate downstream proteins, including troponin. We found that in the presence of ghrelin, cAMP concentrations in cardiomyocytes were lower compared to placebo (first and second columns in Figure 27). Pretreatment of cardiomyocytes with a GHS-R1a antagonist (D-Lys 3) prevented the decrease in cAMP concentrations, remaining similar to placebo treatment (third column in Figure 27). These results are consistent with the ghrelin-mediated decrease in serine 23-24 phosphorylation of troponin I, suggesting that a cAMP-dependent phosphokinase may be responsible for serine 23-24 phosphorylation. Consideration

[0329] This randomized, placebo-controlled trial of intravenous acylated ghrelin versus placebo in patients with advanced CHF and reduced EF achieved the primary outcome of CO2 change. Ghrelin increased CO2 without adverse effects on hypotension, arrhythmias, or ischemia. This was due to a significant increase in SV, with no change in HR. The increase in CO2 was associated with unchanged calculated resting VO2, a decrease in AVO2 difference, an increase in SVO2, and a small but significant decrease in SpO2, suggesting improved O2 delivery to metabolic tissues due to increased CO2 (despite a slight decrease in arterial O2 content measured as SpO2). In vitro data suggest that the increase in CO2 is secondary to a novel mechanism involving inhibition of cAMP production, decreased troponin I phosphorylation, and calcium sensitization. This mechanism, distinct from traditional inotropic mechanisms (which have adverse side effects), may account for the absence of ghrelin-induced side effects.

[0330] Physiological and vascular functions of ghrelin The concentrations and effects of ghrelin have been investigated in normal human subjects (Akamizu et al., 2004; Arvat et al., 2001; Broglio et al., 2001; Broglio et al., 2003a; Broglio et al., 2003c; Broglio F., 2001; Enomoto et al., 2003; Falken et al., 2010; Levin et al., 2006; Nagaya et al., 2001a; Okumura et al., 2002; Peino et al., 2000; Vestergaard et al., 2007a; Vestergaard et al., 2007b; Vestergaard et al., 2008; and Vestergaard et al., 2007c), as well as in patients with cancer cachexia (Garcia et al., 2013; and Garcia et al., 2005), chronic obstructive pulmonary disease cachexia (Miki et al., 2012), growth hormone deficiency (Miki et al., 2012), obesity (Tassone et al., 2003), metabolic syndrome (Tesauro et al., 2005), and Cushing's syndrome (Leal-Cerro et al., 2002).

[0331] Effects have been variable and inconsistent. Small human studies suggest that ghrelin may improve cardiac output (Nagaya et al., 2001b) and left ventricular EF (Nagaya et al., 2004), but these studies did not specify whether the acylated or des-acylated form of ghrelin was used or relate it to specific symptoms of AHF. Furthermore, because acyl and des-acyl forms have different binding and effects on cardiomyocytes (Lear et al., 2010), it is unclear which form of ghrelin is responsible for the observed effects in these studies.

[0332] Potential underlying mechanisms As with many drugs currently in use or under development for chronic and acute heart failure (e.g., beta-blockers, angiotensin receptor-neprilysin inhibitors, levosimendan, serelaxin, uralatide, omecamtib / mecavir, nitroxyl donor, istaloxime, etc.), the mechanisms of action that confer clinical benefit are unclear and likely multifactorial.

[0333] The growth hormone secretagogue receptor (GHSR) is a seven-transmembrane G protein-coupled receptor with multiple downstream signaling pathways. The receptor can heterodimerize with other G protein-coupled receptors, resulting in Gs, Gi, and possibly Gq effects. Four (possibly five) distinct ghrelin receptors with poorly understood functions are present in the heart and vasculature.

[0334] The aforementioned ghrelin signaling and possible mechanisms responsible for the observed inotropic (contractile) effects of ghrelin include the following: 1) GHSR-1a → binds Gaq11 → phospholipase C (PLC) → inositol triphosphate (IP3) and diacylglycerol (DAG) → IP3 receptors on the surface of the endoplasmic (and potentially sarcoplasmic) reticular tissue → release of intracellular calcium (Albarran-Zeckler and Smith, 2013). 2) DAG → activation of protein kinase C (PKC) → activation of voltage-dependent L-type Ca channels → increase in intracellular calcium (Sun et al., 2010b) 3) GHSR → mediated by PLC and protein kinase S (or PKC?) → inhibition of potassium release → prolonged potency → activation of L-type calcium channels (Sun et al., 2010a)

[0335] However, all of these mechanisms result in an unobserved increase in intracellular calcium concentration. It is the increase in intracellular calcium concentration that induces severe side effects in AHF patients. Instead, we found an increase in contractility and fractional shortening secondary to increased calcium sensitivity.

[0336] Our data support a novel mechanism as follows: 4) GHSR → Gi → Decrease in cAMP → Decrease in PKA activity → Decrease in phosphorylation of proteins (e.g., troponin I) → Ca 2+ Changes in sensitivity

[0337] If ghrelin acts via an increase in calcium concentration, it would be potentially associated with the adverse effects of β-adrenergic receptor antagonists and phosphodiesterase inhibitors, which would be counteracted by ghrelin and produce a central sympatholytic effect (Nagaya et al., 2001b). However, as shown below, our data support a model of increased contractility via calcium sensitization. Therefore, if ghrelin functions as an inotropic agent, side effects and adverse events would be expected, but none were observed. This is novel and unexpected, as demonstrated by our in vitro experiments.

[0338] Ghrelin also has many additional potential cardioprotective effects, such as reduced inflammation (Li et al., 2004) and apoptosis (Baldanzi et al., 2002), improved endothelial function by improving nitric oxide bioavailability (Tesauro et al., 2005), and increased lean body mass (Nagaya et al., 2004). Ghrelin has also been observed to have vasodilatory effects, which are often limiting inotropic agents and lead to hypotension. The primary potential benefit of ghrelin observed in this study is its lack of effects on blood pressure and its lack of hypotension.

[0339] conclusion In patients with advanced HF and reduced EF, a 120-minute intravenous ghrelin infusion improved cardiac output compared with placebo without causing adverse hypotension, arrhythmias, tachycardia, or ischemia. Our ex vivo studies in mouse cardiomyocytes suggested that the mechanism may be related to calcium sensitization. These studies demonstrate that ghrelin is effective and safe for the treatment of AHF.

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Claims

1. A pharmaceutical composition for the treatment of acute heart failure (AHF) in an individual, comprising an acylated ghrelin molecule.

2. Use of an acylated ghrelin molecule for the manufacture of a medicament for the treatment of acute heart failure (AHF) in an individual.

3. 2. The pharmaceutical composition of claim 1, wherein the AHF is selected from the group consisting of acute decompensated heart failure (ADHF); hypertension-associated AHF; tachycardia-mediated AHF; pulmonary edema-associated AHF; cardiogenic shock AHF; and severe cardiogenic shock AHF.

4. 4. The pharmaceutical composition of claim 1 or 3, wherein the AHF is not associated with myocardial infarction and / or the AHF is not de novo AHF.

5. Following administration of the acylated ghrelin molecule, the individual may experience increased cardiac output, increased cardiac contractility, increased cardiac stroke volume, improved ventricular function, increased ventricular ejection fraction, decreased phosphorylation of troponin I, increased calcium sensitivity, decreased intracellular cAMP, improved renal function, increased estimated glomerular filtration rate (eGFR), improved dyspnea, improved edema, decreased biomarkers, decreased hypotension, resolution of cardiogenic shock, decreased dizziness, decreased lightheadedness, increased arterial oxygen (AVO) levels, and / or decreased vasoconstriction. 2 5. The pharmaceutical composition of claim 1, 3, or 4, wherein the composition exhibits one or more parameters selected from the group consisting of a decrease in pulmonary artery pressure gradient, an increase in pulmonary blood flow (PBF), a decrease in estimated systemic vascular resistance (eSVR), a decrease in pulmonary capillary wedge pressure, a decrease in left ventricular end-diastolic pressure, a decrease in left ventricular end-diastolic volume, a decrease in pulmonary artery pressure, and a decrease in central venous pressure.

6. The pharmaceutical composition of any one of claims 1 and 3 to 5, wherein the AHF is associated with one or more factors selected from the group consisting of spontaneous worsening of chronic heart failure, infection, allergic reaction, thrombosis, surgery, cardiovascular disease, pulmonary disease, cardiomyopathy, sleep apnea, alcohol use, illegal drug use, anemia, dyslipidemia, hyperthyroidism, Paget's disease, hypertension, prescription drug use, smoking, high blood pressure, renal dysfunction, diabetes, congenital heart disease, lifestyle choices, arrhythmia, tachycardia, bradycardia, inflammation, toxins, autoimmune disease, infiltrative disease, connective tissue disease, metabolic disease, endocrine disease, aging, inherited gene mutation, and pregnancy.

7. 7. The pharmaceutical composition of any one of claims 1 and 3 to 6, wherein the AHF comprises one or more symptoms selected from the group consisting of chest pain, cough, shock, hypertension, oliguria, anuria, fatigue, shortness of breath, hypoxemia, rapid breathing, tachycardia, ischemia, edema, renal dysfunction, hypotension, organ failure, cold extremities, quadriplegia, muscle fatigue, nausea, vomiting, weight loss, pulmonary edema, lower body discomfort, peripheral swelling, hypoperfusion, lower body swelling, cardiac swelling, weight gain, weight loss, cachexia, bulging neck vessels, hepatomegaly, dizziness, syncope, altered mental status, loss of appetite, hypotension, cardiac arrhythmia, difficulty sleeping, discomfort when sitting flat, and sleep apnea.

8. 8. The pharmaceutical composition of any one of claims 1 and 3-7, wherein the individual is diagnosed as suffering from AHF using one or more procedures from the group consisting of x-ray examination, blood test, electrocardiogram (ECG), tracing the individual's medical history, positron emission tomography (PET) scan, multi-gated acquisition (MUGA) scan, scintigraphy, echocardiogram, angiography, hemodynamic measurements, computed tomography (CT) scan, medical examination of symptoms, biomarker measurements, and magnetic resonance imaging (MRI) scan.

9. 9. The pharmaceutical composition of any one of claims 1 and 3 to 8, wherein after administration of the pharmaceutical composition, the individual does not exhibit one or more parameters from the group consisting of increased heart rate, tachycardia, decreased blood pressure, hypotension, increased oxygen requirement, ischemia, increased plasma troponin T, cardiac arrhythmias, and affected calcium transients.

10. The pharmaceutical composition of any one of claims 1 and 3 to 9, wherein the acylated ghrelin molecule comprises one or more of the group consisting of wild-type acylated ghrelin and an acylated ghrelin fusion molecule with wild-type acylated ghrelin.

11. The pharmaceutical composition of any one of claims 1 and 3 to 10, wherein the acylated ghrelin molecule comprises one or more of the group consisting of a synthetic acylated ghrelin molecule, a recombinant acylated ghrelin molecule, and an endogenous acylated ghrelin molecule.

12. The pharmaceutical composition of any one of claims 1 and 3 to 11, wherein the pharmaceutical composition is administered one or more times daily.

13. The pharmaceutical composition of any one of claims 1 and 3 to 12, wherein the pharmaceutical composition is administered by injection.

14. The pharmaceutical composition of any one of claims 1 and 3 to 12, wherein the individual is 18 years of age or older.

15. The pharmaceutical composition according to any one of claims 1 and 3 to 14, wherein the pharmaceutical composition is administered before, during and / or after surgery.

16. The pharmaceutical composition of any one of claims 1 and 3 to 13, wherein the individual is administered with one or more additional therapeutic agents.

17. The pharmaceutical composition of any one of claims 1 and 3 to 16, wherein the pharmaceutical composition comprises one or more additional therapeutic agents.

18. 18. The pharmaceutical composition of claim 16 or 17, wherein the therapeutic agent is one or more therapeutic agents selected from the group consisting of angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, vasopressin receptor antagonists, beta-blockers, inotropic vasodilators, omecamtib mecarbil, renin antagonists, relaxin, ularitide, digoxin, vasodilators, angiotensin II receptor antagonists, aspirin, statins, antihypertensive agents, calcium sensitizers, ivabradine, diuretics, vasopressors, adenosine antagonists, and aldosterone antagonists.

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