TREATMENT OF ACUTE HEART FAILURE
20230126295 · 2023-04-27
Assignee
Inventors
Cpc classification
A61P9/04
HUMAN NECESSITIES
A61K38/25
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61B5/029
HUMAN NECESSITIES
A61B5/02028
HUMAN NECESSITIES
A61B5/318
HUMAN NECESSITIES
International classification
Abstract
The invention provides a ghrelin molecule for use in the treatment and/or prophylaxis of Acute Heart Failure (AHF) in an individual, as well as corresponding methods and uses. The invention further provides associated compositions and kits of parts, for use in the treatment and/or prophylaxis of AHF in an individual.
Claims
1-2. (canceled)
3. A method for the treatment of Acute Heart Failure (AHF) in an individual, the method comprising the step of administering to the individual an acylated ghrelin molecule.
4. (canceled)
5. The method according to claim 3, wherein the AHF is selected from the group comprising: Acute Decompensated Heart Failure (ADHF); AHF associated with hypertension; tachycardia-mediated AHF; AHF associated with pulmonary edema; cardiogenic shock AHF; or severe cardiogenic shock AHF.
6. The method according to claim 5, wherein the Acute Decompensated Heart Failure (ADHF) is Acute Decompensated Chronic Heart Failure (ADCHF) or congestive ADHF, preferably Acute Decompensated Chronic Heart Failure (ADCHF).
7-9. (canceled)
10. The method according to claim 3, wherein the AHF is not associated with a myocardial infarction.
11. The method according to claim 3, wherein the AHF is not de novo AHF.
12. The method according to claim 3, wherein after administration of the ghrelin molecule the individual exhibits one or more parameters from the group comprising: an increased heart output; and/or an increased heart contraction; and/or an increased heart stroke volume; and/or an increased ventricular function; and/or an increased ventricular ejection fraction; and/or reduced troponin I phosphorylation; and/or an increased calcium sensitivity; and/or reduced intracellular cAMP; and/or improved renal function; and/or improved estimated glomerular filtration rate (eGFR); and/or improved dyspnea; and/or improved edema; and/or reduced biomarkers; and/or reduced hypotension; and/or resolution of cardiogenic shock; and/or reduced dizziness; and/or reduced light-headedness; and/or a reduced arterio venous oxygen (AVO2) difference; and/or an increase in pulmonary blood flow (PBF); and/or a reduced estimated systemic vascular resistance (eSVR); and/or reduced pulmonary capillary wedge pressure; and/or reduced left ventricular end-diastolic pressure; and/or reduced left ventricular end-diastolic volume; and/or reduced pulmonary artery pressure; and/or reduced central venous pressure.
13-22. (canceled)
23. The method according to claim 3, wherein the AHF is associated with one or more factors of the group comprising: spontaneous worsening of chronic heart failure; an infection; and/or an allergic reaction; and/or a blood clot; and/or surgery; and/or cardiovascular disease; and/or lung disease; and/or cardiomyopathy; and/or sleep apnea; and/or alcohol consumption; and/or recreational drug consumption; and/or anaemia; and/or dyslipidemia; and/or an overactive thyroid; and/or Paget's disease; and/or hypertension (such as pulmonary hypertension); and/or prescription medication consumption; and/or smoking; and/or high blood pressure; and/or kidney dysfunction; and/or diabetes; and/or congenital heart defects; and/or lifestyle choices; and/or an irregular heartbeat; and/or rapid heartbeat; and/or slow heartbeat; 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 old age; and/or hereditary genetic mutations; and/or pregnancy.
24-25. (canceled)
26. The method according to claim 3, wherein the AHF comprises one or more of the symptoms of the group comprising: chest pain; and/or a cough; and/or shock (such as, cardiogenic shock); and/or high blood pressure; 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, a worsening edema); and/or impaired renal function (such as, worsening renal function); and/or low blood pressure; and/or organ failure (such as liver failure and/or kidney failure); and/or cold extremities; and/or numb extremities; and/or muscle fatigue; and/or nausea; and/or vomiting; and/or weight loss (such as anorexia); and/or pulmonary edema; and/or discomfort of the lower body; and/or peripheral swelling; and/or hypo-perfusion; and/or swelling of the lower body; and/or swelling of the heart; and/or weight gain (such as, sudden weight gain); and/or weight loss; and/or cachexia; and/or elevated neck blood vessels (such as an elevated neck blood vein and/or jugular venous distension); and/or hepatomegaly; and/or dizziness; and/or fainting (also known as syncope); and/or an altered mental state (for example, anxiety and/or confusion and/or depression); and/or loss of appetite; hypotension; and/or arrhythmia; and/or difficulty sleeping; and/or discomfort when lying flat; and/or sleep apnea.
27-30. (canceled)
31. The method according to claim 3, wherein the individual is diagnosed as having AHF using one or more of the procedures of the group comprising: an X-ray; and/or a blood test; and/or an electrocardiogram (BCG); and/or based on the medical history of the individual; and/or a positron emission tomography (PET) scan; and/or multigated acquisition (MUG A) scan; and/or scintigraphy; and/or an echocardiogram; and/or an angiogram; and/or hemodynamic measurement; and/or a computerised tomography (CT) scan; and/or a physical examination of symptoms; and/or measuring biomarkers (such as serum natriuretic peptides and/or plasma natriuretic peptides); and/or, a magnetic resonance imaging (MM) scan.
32-38. (canceled)
39. The method according to claim 3, wherein after administration of the acylated ghrelin molecule the individual does not exhibit one or more parameters of the group comprising: an increased heart rate; and/or tachycardia; and/or a decreased blood pressure; and/or hypotension; and/or an increased oxygen demand; and/or ischemia; and/or increased plasma troponin T; and/or heart arrhythmias; and/or affected calcium transients.
40-49. (canceled)
50. The method according to claim 3, wherein the acylated ghrelin molecule comprises one or more of the group comprising: a modified ghrelin; and/or a ghrelin fusion molecule; and/or a ghrelin fragment; and/or a ghrelin variant; and/or a ghrelin derivative; and/or, wildtype ghrelin.
51. The method according to claim 3, wherein 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.
52. The method according to claim 3, wherein the ghrelin molecule is administered once or more each day, preferably twice each day.
53. The method according to claim 3, wherein the ghrelin molecule is administered by infusion.
54. (canceled)
55. The method according to claim 3, wherein the individual is aged 18 years or older, preferably 65 years or older.
56. The method according to claim 3, wherein the acylated ghrelin molecule is administered before surgery, and/or during surgery, and/or after surgery.
57. The method according to claim 3, wherein the individual is administered with one or more additional therapeutic agents.
58-60. (canceled)
61. The method according to claim 57, wherein the additional therapeutic agent is one or more therapeutic agent selected from the group comprising: angiotensin-converting enzyme (ACE) inhibitors; and/or angiotensin II receptor blockers; and/or vasopressin receptor antagonists; and/or beta blockers; and/or an inodilator (in particular, mirinone and/or enoximone and/or dobutamine and/or levosimendan); and/or omecamtiv mecarbil; and/or renin antagonist; and/or relaxin; and/or ularitide; and/or digoxin (Lanoxin); and/or vasodilators; and/or angiotensin II receptor antagonists (such as valsartan); and/or aspirin; and/or statins; and/or antihypertensive drugs (such as sacubitril); and/or calcium sensitisers; and/or ivabradine; and/or diuretics; and/or vasopressor (such as noradrenaline, dopamine, vasopressin, and/or angiotensin II); and/or adenosine antagonists; and/or aldosterone antagonists.
62-70. (canceled)
Description
[0288] The present invention will now be described with reference to one or more non-limiting figures and example.
[0289]
[0290]
[0291]
[0292]
[0293]
[0294]
[0295]
[0296]
[0297]
[0298]
[0299]
[0300]
[0301]
[0302]
[0303]
[0304]
[0305]
[0306]
[0307]
[0308]
[0309]
[0310]
[0311]
[0312]
[0313]
[0314]
EXAMPLE
[0315] The following example is included to demonstrate particular embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the example which follows represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate 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.
Summary of the Experimental Work
Introduction
[0316] Ghrelin is an endogenous appetite-stimulating peptide hormone with potential cardiovascular actions. It has long been considered that ghrelin is a potential inotrope agent, and assumed that it acts by increasing intracellular Ca.sup.2+ concentrations. Based on this understanding, it was widely expected that ghrelin would have a number of side effects that would be highly dangerous for a patient with acute heart failure (AHF), including increased heart arrhythmias, hypotension and ischemia (Abraham et al., 2005; Cuffe et al., 2002; Mebasaa et al, 2007; and Packer et al., 2013).
[0317] Contrary to that understanding, the inventors have surprisingly identified that ghrelin does not increase intracellular Ca.sup.2+ concentrations, but increases sensitivity to existing Cat. This led the inventors to hypothesize that the previous assumptions of ghrelin acting as a conventional inotrope and thus causing hypotension, ischemia and arrhythmia were incorrect and, contrary to the previous misconceptions regarding ghrelin's function, ghrelin could act as a new type of inotrope and be used to treat AHF without causing hypotension, ischemia or arrhythmia.
[0318] To test this hypothesis, the inventors tested ghrelin on heart failure patients. In order to avoid inadvertently causing harm to patients on which the ghrelin was tested, the inventors conducted a clinical study on patients with advanced heart failure. Advanced heart failure is similar to AHF, but it was expected that the severe side effects that would be observed in AHF in response to an inotrope would not occur to the same extent. Therefore, whilst administering ghrelin to a patient with advanced heart failure would provide a very similar indication of the safety and effectiveness of ghrelin if administered to an AHF patient, the tests could be conducted in a safer manner.
[0319] As explained below, in the clinical study the inventors confirmed that although ghrelin acted as an inotrope it did not increase Ca2.sup.+ concentrations. Most importantly, the inventors demonstrated that ghrelin did not cause any of the side effects that would be seriously problematic for a patient with AHF, such as increased heart arrhythmias, hypotension and ischemia. The experiments also show that treatment with ghrelin leads to an improvement in signs which would also occur in AHF, demonstrating that ghrelin would be effective in the treatment of that disease.
Heart Failure and its Forms and Outcomes
[0320] Heart failure (HF) is defined as inadequate cardiac output to meet metabolic demands or adequate CO only secondary to compensatory neurohormonal activation. 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).
[0321] AHF most often occurs when the chronic compensation is inadequate and is termed acute decompensated HF (ADHF). A majority of this is in patients with pre-existing CHF, thus the term acute decompensated chronic HF, ADCHF.
[0322] Although medical and device therapy have improved outcomes in CHF with reduced ejection fraction (HFrEF), 5-10% of patients suffer advanced (also termed severe or end-stage) CHF, characterized by reduced cardiac output (CO), progressive end organ failure, frequent repeated hospitalizations for ADCHF, and high risk of death.
[0323] In ADHF and ADCHF, in-hospital mortality ranges 5-10%, and median length of hospital stay is 5-8 days. Over 50% of patients are discharged with unresolved symptoms, and within 30 or 60 days in different studies, half have again worsening symptoms, one fourth are re-hospitalized and over 10% have died (Baker et al., 2003; Curtis et al., 2008; Gheorghiade et al., 2006; Go et al., 2014; Polanczyk et al., 2000). Mortality at 1 year in population wide registries is 25-35% (Lund, 2017). After an improvement in outcomes in the late 1990s, prognosis in CHF and ADCHD has not improved since 2000 (Baker et al., 2003; Curtis et al., 2008; Polanczyk et al., 2000; Thorvaldsen et al., 2016). Costs to society for HF are projected to increase 3-fold between 2010 and 2030 and most of this cost is related to ADCHF (Heidenreich et al., 2013).
[0324] Among hospitalizations for ADCHF, about 5% have severe hemodynamic compromise, with hypotension and shock, arrhythmias and global ischemia (even in the absence of obstructive coronary artery lesions), and the short-term mortality in this group exceeds 50%.
Inotrope Therapy for Advanced HF and ADCHF
[0325] Most inotrope drugs work by increasing intracellular Ca.sup.2+ concentrations (e.g. milrinone, dobutamine); some work by increasing the sensitivity of contractile proteins to existing Ca.sup.2+ (levosimendan); but the former increases oxygen demand and all have multiple and complex mechanisms involving vasodilation. In clinical trials and clinical practice, existing inotropes indeed universally cause hypotension, tachy-arrhythmias and ischemia, and have neutral effect on or increase mortality (Abraham et al., 2005; Cuffe et al., 2002; Mebazaa et al., 2007; Packer et al., 2013). Three main classes of inotropes are in use: phosphodiesterase (PD)-inhibitors (e.g. milrinone); adrenergic agonists (e.g. dobutamine) and levosimendan. Specifically, in the randomized OPTIME CHF, milrinone vs. placebo caused significantly more hypotension and atrial arrhythmias and non-significantly more deaths (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 were developed for longer term use but compared to placebo, caused more arrhythmias, vertigo (likely due to hypotension), cardiac death, and sudden death (Amsallem et al., 2005; Cohn et al., 1998; Packer et al., 1991; Uretsky et al., 1990). Intermittent intravenous dobutamine infusions reduced HF hospitalization (Oliva et al., 1999), but in a subsequent study that was stopped early and never published, it increased mortality (Dies et al., 1986) and in another there was no control group and 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 vs. placebo in REVIVE I and II (Packer et al., 2013). Thus, existing inotropes worsen outcomes, yet are sometimes used in severe ADCHF and advanced CHF when organ function is deteriorating, patients cannot be mobilized and discharged from hospital, when bridging to heart transplantation, and/or death is imminent.
Ghrelin
[0326] Ghrelin (“ghre”=grow) was originally identified as a 28 amino acid peptide hormone that is the endogenous ligand for the growth hormone (GH) secretagogue receptor (GHSR), and partially acts by stimulating GH release (Kojima et al., 1999). Ghrelin has received attention mainly as a centrally acting appetite stimulant (Cummings et al., 2002). Ghrelin is released from the stomach in response to fasting and weight loss, whereas release is inhibited by food intake (Kojima et al., 1999; Kojima and Kangawa, 2005; Shiiya et al., 2002). Ghrelin is acylated (“activated”) at amino-acid 3, and the acyl/acylated form is believed to be responsible for most of ghrelin's actions (Hosoda et al., 2000; Soares and Leite-Moreira, 2008).
Ghrelin's Potential Cardiovascular Actions
[0327] Ghrelin is elevated in cachectic (Nagaya et al., 2001c) and non-cachectic CHF (Lund et al., 2009) but there appears to be resistance to the appetite stimulating effects, which resolves after heart transplantation (Lund et al., 2009). Beyond metabolic effects, ghrelin appears to have specific cardiovascular actions. Ghrelin receptors (growth hormone secreatagogue receptors; GHSR) are widely distributed in cardiac and skeletal muscle and endothelium (Papotti et al., 2000). It is possible that ghrelin is elevated in HF as a compensatory response to poor cardiac function, analogously to compensatory elevations of catecholamines and natriuretic peptides, and indeed acylation of ghrelin is increased in HF, potentially representing an adaptive compensatory response, and decreases post heart transplantation (Zabarovskaja et al., 2014).
Previous Data on Ghrelin Treatment
[0328] In rat HF models, ghrelin increased CO and fractional shortening (Nagaya et al., 2001d); in rat myocardial infarction models, ghrelin reduced cardiac sympathetic 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 suggested ghrelin may improve cardiac output (Nagaya et al., 2001b) and left ventricular ejection fraction (EF), exercise capacity and muscle wasting (Nagaya et al., 2004) but these studies do not specify whether the ghrelin form used was acylated or not. The cardiovascular actions of ghrelin have been reviewed, but data are inconsistent, showing both positive and negative inotropic effects (Soares et al., 2005), and refer to variable forms of ghrelin and predominantly the non-acylated inactive form (Broglio et al., 2003b; Isgaard, 2013; Kishimoto et al., 2012; Leite-Moreira et al., 2008; Nagaya and Kangawa, 2003a, b, 2006; Nagaya et al., 2006). Furthermore, the safety, clinical efficacy and mechanisms of action of ghrelin in HF are unknown. GHSR agonists such as pralmorelin and hexarelin have fewer amino acids and no sequence similarity. Pralmorelin but not ghrelin or hexarelin improved CO in dogs with acute myocardial infarction (US patent US2004014671A1).
Present Aims
[0329] Based on the 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 underlying mechanism for any clinical effect, we also assessed contractility and cellular Ca.sup.2+ transients in response to ghrelin in isolated cardiomyocytes from healthy control and HF mice.
Methods Human Trial
Study Design and Setting
[0330] Between 17 Feb. 2013 and 19 May 2015 we conducted a prospective double-blind, placebo-controlled, parallel-group, single centre randomized clinical trial with a one-time treatment of intravenous acyl ghrelin or placebo.
Patients
[0331] Patients were pre-screened in the Karolinska University Hospital heart failure clinic (
TABLE-US-00002 TABLE 1 Inclusion and exclusion criteria Inclusion criteria 1. Heart failure defined as symptoms and signs of heart failure in judgement of investigator 2. Left ventricular ejection fraction (EF) at screening and on day of treatment of ≤40% 3. New York Heart Association (NYHA) class III or IV 4. Written informed consent Exclusion criteria 1. Age <18 2. Current smoker that cannot refrain from smoking from day of treatment 3. Consumption of caffeine on day of treatment 4. Current alcohol or drug abuse 5. Acute coronary syndrome in last 3 months 6. Received heart transplantation or left ventricular assist device ever 7. Oxygen dependent lung disease 8. Peripheral saturation by pulse oximetry <95% on study day 9. Creatinine clearance (Cockgroft-Gault) or estimated glomerular filtration rate (eGFR, by MDRD) <30 mL/min on study day 10. Dementia or inability to give informed consent 11. Any solid organ transplant 12. Current hormonal treatment 13. Current immunosuppressive treatment other than corticosteroids 14. Any inotropes within two weeks prior to study day 15. Any gastrointestinal surgery except appendix, gall bladder, hernia, colon. 16. Current gastrointestinal disease other than reflux disease or dyspepsia 17. Known cirrhosis 18. Systolic blood pressure <90 mmHg on study day Blood glucose >20 mmol/L after breakfast on study day that cannot be reduced to <10 with insulin
Preparation of Ghrelin
[0332] Ghrelin is a 28 amino acid peptide with a molecular weight of 3,371 g/mol. Half-time in plasma is 24-30 minutes. Concentrations in normal humans has variably been reported at 100-300 pmol/L (300-900 ng/L) for total ghrelin. There is limited data on acyl ghrelin but we recently measured acyl ghrelin in 41 healthy non-obese adults: fasting: mean±SD: 118±14 ng/L, 5-95th percentile 27-328 ng/L; trough 60 minutes post 260 kcal mixed meal: 80±12 ng/L, 5-95th percentile 21-293 ng/L. Synthetic acylated (active) human ghrelin (brand name Clinalfa Ghrelin (human) 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 (Tokyo, Japan). For each patient and treatment, a stock solution was prepared consisting of multiple vials of powder ghrelin (100 μg ghrelin/vial together with phosphate buffer), each vial dissolved in 1 mL sterile water for infusion (B. Braun, Germany) and visually inspected to be a clear and colorless solution, prior to adding 0.001 g (0.02 ml of 50 g/L stock) human albumin (for concentration 0.001 g/mL=0.1%) to each vial and further mixed with normal saline (NaCl 9 mg/ml, B. Braun, Germany). The proportion of stock solution and saline were according to patient weight (Table 2), resulting in a total volume of 100 mL (to ensure the same volume infusion for all patients). The final intravenous infusion rate was 0.50 mL/min volume (total volume 60 mL), equivalent to 0.1 μg (30 pmol)/kg/min (total amount 12.0 μg/kg) of acyl ghrelin administered to all patients randomized to ghrelin. The infusion was given for 120 minutes and continued until all measurements at 120 minutes had been completed (thus delivering some additional ghrelin). The median total infusion duration was 171 minutes. Measurements were repeated 30 minutes after stopping infusion.
TABLE-US-00003 TABLE 2 Stock solution, NaCl and final volume Total Total acyl Stock solution Infusion solution volume ghrelin acyl Volume Amount Volume acyl (ml) (ug) Patient ghrelin of stock acyl of ghrelin Infused infused weight concentration solution ghrelin NaCl concentration over 120 over (kg) (ug/ml) (ml) (ug) (ml) (ug/ml) min* 120 min 50 100 10 1000 90 10 60 600 55 100 11 1100 89 11 60 660 60 100 12 1200 88 12 60 720 65 100 13 1300 87 13 60 780 70 100 14 1400 86 14 60 840 75 100 15 1500 85 15 60 900 80 100 16 1600 84 16 60 960 85 100 17 1700 83 17 60 1020 90 100 18 1800 82 18 60 1080 95 100 19 1900 81 19 60 1140 100 100 20 2000 80 20 60 1200 105 100 21 2100 79 21 60 1260 110 100 22 2200 78 22 60 1320 115 100 23 2300 77 23 60 1380 120 100 24 2400 76 24 60 1440 125 100 25 2500 75 25 60 1500 130 100 26 2600 74 26 60 1560 135 100 27 2700 73 27 60 1620 140 100 28 2800 72 28 60 1680 145 100 29 2900 71 29 60 1740 150 100 30 3000 70 30 60 1800 *The last data collection during infusion occurred at 120 minutes. The infusion was stopped after all data had been collected, at a median time of 171 minutes after start of infusion.
Preparation of Placebo
[0333] A solution of physiological normal saline (NaCl 9 mg/mL, B. Braun, Germany) was infused at the same volume and rate (0.50 mL/min) and total duration as the ghrelin infusion.
Procedures And Data Collection
[0334] Patients potentially eligible during pre-screening and providing written informed consent reported to the laboratory at 8:00 am in the fasting state. Examinations are shown schematically in
[0335] Patients were randomized in parallel by block randomization in groups of 4 using paper envelopes to ghrelin or placebo and the infusion was given by antecubital vein over 120 minutes. A dedicated study nurse prepared ghrelin and placebo for infusion and patients and all other investigators were blinded. Immediately prior to, during, and after the 120 minute infusion and also 30 minutes after stopping the infusion, blood samples were drawn and symptoms, signs, echocardiography, ECG, and non-invasive hemodynamics were assessed (
Pre-Specified Trial Efficacy Outcomes
[0336] The primary efficacy outcome was difference between ghrelin and placebo in change in CO from start of infusion (time 0) to end of infusion (time 120 minutes). Numerous secondary outcomes related to hemodynamics, echocardiography findings and plasma biomarkers in response to the 120 minute infusion, 30 minutes after stopping the infusion and 2-5 days after stopping the infusion were also assessed (see results).
Pre-Specified Trial Safety Outcomes
[0337] Safety outcomes included reduction in systolic blood pressure, hypotension, and symptomatic hypotension, prolongation of the QTc, ischemia, and arrhythmia during or after infusion, and clinical outcomes.
Data Collection Methods and Definitions
Cardiac Output and Hemodynamics
[0338] Non-invasive resting CO in was assessed in duplicate at each measurement using the Innocor® device (Innovision, Odense, Denmark). The Innocor is a non-invasive device that measures pulmonary blood flow using an inert gas rebreathing technique, and measures VO.sub.2 directly. It has been validated for CO and VO.sub.2 at rest and exercise (Gabrielsen et al., 2002; Stahlberg et al., 2009). The coefficient of variance is low (VO.sub.2<2%; CO 5-7%)(Stahlberg et al., 2009), similar to the gold standard Fick method (Warburton et al., 1999) and superior to other non-invasive methods (Warburton et al., 1999). In the absence of a significant intrapulmonary shunt, pulmonary blood flow as calculated by inert gas rebreathing measured by the Innocor® has been shown to provide a reliable estimate of CO (Stahlberg et al., 2009).
[0339] The Innocor® directly measures pulmonary blood flow, VO.sub.2 and SpO.sub.2. From these variables, shunt fraction, cardiac output, SVO.sub.2 and A-V O.sub.2 difference was then calculated using standard formulas. Beat-by-beat ECG, blood pressure and the first derivative of the pressure signal (+dP/dt) were measured using a Nexfin® device (described below). Fifteen minutes of continuous beat-by-beat Nexfin data were averaged at the time of Innocor measurements. Estimated systemic vascular resistance (eSVR) was subsequently calculated as mean blood pressure/cardiac output×80. Stroke volume (ml) was calculated as Innocor derived cardiac output/Nexfin derived heart rate×1000.
Continuous Hemodynamic and EKG Monitoring
[0340] A plethysmographic based (finger-cuff) approach was used to measure blood pressure (Nexfin®, BMEYE B.V., Amsterdam, The Netherlands). Nexfin was also used for heart rate monitoring. Heart rate and blood pressure data were measured continuously, beat-by-beat, throughout the study protocol. The data was then averaged over 15 minutes following the Innocor measurements. In addition EKG was assessed before start of and during infusion at 60 and 120 minutes and after the ad lib lunch. QT intervals were measured in the precordial lead V5 in 12-lead surface ECGs, at 50 mm/sec paper speed and 10 mm/mV amplitude. Heart rate corrected QT intervals (QTc) were manually calculated with the Bazett formula, QTc=QT/RR.sup.1/2, using the RR interval preceding the measured QT interval. The mean QTc from 3 heart beats was recorded.
Echocardiography
[0341] Echocardiography was performed by a technician blinded to treatment allocation and all echo images were analyzed and interpreted by one independent investigator blinded to treatment allocation and clinical history of the patients. Two dimensional images were recorded with a Vivid 7/E9 ultrasound system (GE, Horten, Norway) with a 3 MHz Doppler transducer. A detailed echocardiographic examination including dimensions, cardiac systolic and diastolic function, valve performance and systolic pulmonary artery pressure was performed at baseline (Table 5). A shorter protocol including left ventricular dimensions and function, stroke volume and cardiac output was used for comparison of changes over time (Table 9 and 10). Left ventricular end-systolic and end-diastolic volumes and left ventricular ejection fraction (LVEF) were measured using the modified biplane Simpson's method. LVEF was also measured using the Teichholz method. Because of missing data on LVEF by the Simpson's method due to low image quality, changes in LVEF over time was calculated using the Teichholz method. Left atrial volumes were calculated using the biplane area length method from the apical four- and two-chamber views and indexed to the body surface area. The E/e′ ratio was calculated using the peak E-wave velocity of the mitral inflow and the averaged of septal and lateral tissue Doppler recordings. Tricuspid annular peak systolic excursion (TAPSE) was assessed with M-mode echocardiography. Speckle tracking was used for left ventricular longitudinal strain. Changes in strain over time was calculated using the average of the regional strain from septal and inferior segments. 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 the echo derived stroke volume×heart rate.
TABLE-US-00004 TABLE 5 Baseline echocardiography data. P values by Wilcoxon rank sum test for continuous variables and by Fischer’s exact test for categorical variables. Continuous variables are presented as medians (IQR) and categorical variables as %. Missing p Variable n (%) Ghrelin Placebo value LVEDD (mm) 2 (7) 69 (60-75) 67 (61-73) 0.878 LVESD (mm) 2 (7) 63 (53-67) 60 (51-66) 0.983 LVESV (ml) 14 (45) 145 (92-218) 139 (50-176) 0.242 LVEDV (ml) 14 (45) 206 (163-304) 197(100-244) 0.329 Septum (mm) 2 (7) 10 (8-12) 11 (9-13) 0.367 Posterior wall 2 (7) 7 (7-8) 8 (7-10) 0.346 (mm) LV mass (g) 2 (7) 248 (194-337) 293 (231-377) 0.183 LVEF Simpson 14 (45) 28 (25-41) 30 (24-36) 0.922 (%) LVEF visual (%) 1 (3) 30 (20-35) 20 (20-33) 0.192 LVEFTeicholz 2 (6) 29 (20-34) 19 (13-33) 0.169 (%) SV (ml) 2 (6) 44 (39-76) 56 (40-67) 0.896 CO (mL/min) 2 (6) 3560 3560 (2930- 0.760 (30904660) 4340) LAV (ml) 2 (7) 90 (70-141) 120 (75-155) 0.325 LAVi (ml/m.sup.2) 2 (7) 47 (34-63) 53 (41-67) 0.513 RA area (cm.sup.2) 1 (3) 25 (21-30) 26 (19-31) 0.852 MR ≥2, n (%) 1 (3) 1 (7) 0 1.000 TR ≥2, n (%) 1 (3) 1 (7) 2 (13) 0.179 AR ≥2, n (%) 1 (3) 1 (7) 0 0.651 MS, n 0 0 AS, n 0 0 TAPSE (mm) 1 (3) 13 (10-17) 11 (6-16) 0.271 SPAP (mmHg) 8 (25) 39 (33-50) 30 (30-45) 0.304 E/e′ sept 1 (3) 17 (13-22) 20 (14-28) 0.281 E/e′ lateral 1 (3) 13 (9-17) 12 (8-18) 0.917 e' septal (cm/s) 0 5 (4-6) 5 (3-6) 0.345 e' lateral (cm/s) 0 8 (4-9) 8 (5-11) 0.567 Abbreviations: LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter, LVESV, left ventricular end systolic volume; LVEDV, left ventricular end diastolic volume; LEVF, left ventricular ejection fraction; SV, stroke volume; CO, cardiac output; LV mass, left ventricular mass; LAV, left atrial volume; LAVi, left atrial volume index; RA, right atrial; MR, mitral regurgitation; TR, tricuspid regurgitation; AR, aortic regurgitation; MS, mitral stenosis; AS, aortic stenosis; TAPSE, tricuspid annular plane systolic excursion; SPAP, systolic pulmonary artery pressure; E, E-wave mitral inflow; E/e′, E-wave/e′ ratio; e′, mitral relaxation velocity.
TABLE-US-00005 TABLE 9 Acute echocardiography effects of ghrelin vs. placebo Ghrelin Placebo INF + INF + 120 INF + INF + P- Variable BL 60 min min +30 min BL 60 min 120 min +30 min interaction LVEDD 66 ± 9 67 ± 9 67 ± 10 65 ± 9 69 ± 11 68 ± 11 68 ± 11 69 ± 11 0.70 (mm) LVESD 58 ± 9 58 ± 11 57 ± 11 56 ± 11 61 ± 12 61 ± 14 60 ± 13 61 ± 13 0.98 (mm) LVEF 27.4 ± 7.4 29.1 ± 11.5 29.8 ± 11.4 30.1 ± 11.2 26.7 ± 12.2 24.9 ± 15.4 25.4 ± 13.2 25.5 ± 13.1 0.29 (%) TAPSE 13.0 ± 5.9 13.1 ± 4.8 14.2 ± 5.4 14.1 ± 5.4 11.7 ± 5.0 9.8 ± 3.8* 10.7 ± 5.6 11.1 ± 4.5 0.075 (mm) Strain 11.9 ± 6.3 13.4 ± 4.6 13.2 ± 5.8 12.46 ± 4.1 11.4 ± 8.1 11.6 ± 5.8 11.4 ± 7.6 11.8 ± 9.3 0.51 (%).sup.† E/e′ 15.2 ± 5.5 17.1 ± 9.5 16.3 ± 8.9 15.3 ± 6.6 18.1 ± 6.7 19.8 ± 8.2 18.2 ± 5.9 19.8 ± 5.6 0.97 (ratio) SV 54.3 ± 18.4 56.3 ± 21.4 59.5 ± 25.5 62.2 ± 23.8 59.1 ± 23.3 49.8 ± 16.6* 50.7 ± 13.5* 52.6 ± 19.3 0.021 (ml) CO 3.8 ± 1.5 3.9 ± 1.3 3.9 ± 1.4 4.1 ± 1.5 4.1 ± 2.0 3.4 ± 1.0 3.5 ± 1.0 3.6 ± 1.3 0.12 (L/min) * = p < 0.05 vs. baseline within treatment group .sup.† = n = 13 in ghrelin group and 10 in placebo group
TABLE-US-00006 TABLE 10 Echocardiography at baseline and at 2-5 day follow-up Ghrelin Placebo p- 2-5 days 2-5 days inter- Variable Baseline FU Baseline FU action LVEDD 66 ± 9 66 ± 10 66 ± 9 69 ± 11 0.82 (mm) LVESD (mm) 58 ± 9 57 ± 11 61 ± 12 61 ± 10 0.90 LVEF (%) 27.4 ± 7.4 28.5 ± 11.9 26.7 ± 12.2 24.9 ± 14.7 0.62 TAPSE (mm) 13.0 ± 5.9 11.8 ± 6.0 11.7 ± 5.0 11.3 ± 4.8 0.81 Strain (%).sup.†† 11.9 ± 6.3 10.4 ± 6.4 11.4 ± 8.1 10.3 ± 5.7 0.93 E/e′ (ratio) 15.2 ± 5.5 16.2 ± 8.3 18.1 ± 6.7 16.1 ± 2.8 0.37 SV (ml) 54.3 ± 18.4 52.5 ± 20.6 59.1 ± 23.3 50.43 ± 17.9 0.52 CO (L/min) 3.8 ± 1.5 3.8 ± 1.3 4.1 ± 2.0 3.6 ± 1.3 0.52 Longer term effects comparing baseline (before infusion starts) vs. follow-up approximately 2-5 days after infusion. * = p < 0.05 vs. baseline within group. .sup.† = p < 0.05 ghrelin vs. placebo at same time point. .sup.†† = n = 12 in ghrelin group and 10 in placebo group
Symptoms and Signs
[0342] The following symptoms were assessed as yes or no prior to infusion and after 30, 60, 120 min and 30 min after infusion and at the 2-5 days follow-up; headache, dizziness, dyspnea, central chest pain, flush, sleepiness, upset stomach and other symptoms. The following signs were recorded prior to start of infusion and after lunch; rales, peripheral oedema, jugular venous distension, hepatomegaly and S3 gallop.
Blood Samples
[0343] Blood samples were collected in 1) morning fasting state baseline, 2) after standardized breakfast prior to intervention, 3) after 30 min infusion, 4) after 60 min infusion and 5) after 120 min infusion, 6) 30 min after completed infusion and 7) at follow-up 2-5 days later (
[0344] In samples dedicated for ghrelin measurements a protease inhibitor cocktail was prepared, consisting of 5.5 μl 10 mM KR-62436 (DPP4 inhibitor) in DMSO and a SIGMAFAST® protease inhibitor tablet (both produced by Sigma-Aldrich Corp., St. Louis, Mo., USA) dissolved in 2100 μl of distilled water (50×stock). Blood samples were drawn using 6 mL EDTA plasma tubes, immediately put on ice and 160 μl of the 50×protease inhibitor cocktail was added. Tubes were vortexed for 10 seconds and centrifuged at 4° C., 10 min, 2500 relative centrifugal force (RCF, or g). The resulting supernatant (plasma) was then pipetted into Eppendorf tubes (450 μl each) and immediately frozen at −70° C. until analyzed.
Serum and Plasma Biomarkers
[0345] Creatinine clearance was calculated according to the Cockcroft-Gault equation; ([140-age] * weight in kg*1.23)/creatinine*0.85 (if woman). Renal function was measured by creatinine and cystatin-C at Karolinska University Hospital core laboratory. NT-proBNP was analyzed by proBNPII (Roche Diagnostics, Bromma, Sweden). Plasma glucose measurements were from EDTA-containing whole venous blood tubes and analyzed with a photometric point-of-care technique, the HemoCue® Glucose 201 RT (Ängelholm, Sweden). Troponin T was analyzed in the Karolinska University Hospital core laboratory.
Acyl Ghrelin and Pharmacokinetics
[0346] Plasma concentrations of active (acylated) ghrelin were assayed by a custom ELISA using electrochemiluminescence detection. Plasma samples were thawed and vortexed. They were then analyzed in duplicate on 96-well multispot plates (Meso Scale Diagnostics, Rockville, Md., USA) coated with capture antibodies against acyl-ghrelin according to manufacturer instructions. The Meso Scale Diagnostics Sector Imager 2400 was used to read the plates. The coefficients of variation (CV %) calculated from plasma concentrations after curve fitting were 9.3/3.5 (intra/inter-assay CV %). The lower limit of detection was 3.2 ng/L and upper limit was set at 10,000 ng/L.
Safety Outcomes
Hypotension
[0347] Hypotension was assessed in two ways: The difference in change in blood pressure between the groups was quantified, and hypotension in any one patient was defined as a drop of systolic blood pressure of >20 mm Hg or to <80 mm Hg.
Arrhythmias
[0348] The difference in change in heart rate between the groups was quantified. Bradycardia was defined as a reduction in heart rate of >15 beats per minute, to a heart rate <40 beats per minute, an increase in PQ time by >20 ms, or AV block II-III. In individual patients, tachycardia was defined as an increase in heart rate by >10 beats per minute or heart rate >110 beats per minute. Ventricular extraystoles were quantified and ventricular tachycardia was defined as non-sustained if ≥3 beats in a row up to ≤30 seconds, and sustained if >30 seconds. Supra-ventricular arrhythmia was defined as ectopic atrial tachycardia, atrial flutter or atrial fibrillation, or AV-nodal tachycardia of >3 seconds duration. Ghrelin may activate hERG channels and as such cause a prolongation of the QTc interval on the EKG, which theoretically could predispose to arrhythmia. Therefor we monitored QTc.
Ischemia
[0349] Ischemia was assessed in two ways: The difference in change in troponin T between the groups was quantified, and ischemia in any one patient was defined as symptoms of ECG changes consistent with ischemia, or a rise in troponin T of >50%.
Clinical Outcomes
[0350] Patients were followed in the medical record until 30 Apr. 2017. Outcomes were freedom from all-cause death; all cause death or HF hospitalization; all cause death or heart transplantation or left ventricular assist device, and a combination of all these.
Statistics
[0351] For baseline characteristics, continuous variable were compared by the Wilcoxon rank sum test for continuous variables and by Fischer's exact test for categorical variables.
[0352] In order to compare the effect of ghrelin vs. placebo on continuous variable over time, a 2-way repeated measures ANOVA (2W-RM-ANOVA) was used to evaluate treatment effect within treatment groups. The 2W-RM-ANOVA has two factors: treatment (2 levels: ghrelin or placebo) and time (3 levels: baseline, after 60 minutes infusion, and after 120 minutes infusion). The main analysis to be considered in this statistical model is the interaction (treatment×time) where we test the significance of the null-hypothesis that the differences between treatments are the same at all time points. Time factor makes little sense in this analysis since we expect a time effect. Treatment factor makes little sense in this analysis since we do not expect a ghrelin vs. placebo group difference at baseline. Post-hoc testing,
[0353] Tukey adjusted p-values for repeated measures within each treatment group and Sidak between treatment groups were performed to test for significant changes. We also assessed differences between the treatment groups at 60 and 120 minutes using unpaired t-tests of absolute and % delta values at 60 and 120 min time points after infusion start within groups by Wilcoxon sign-rank test and between groups by Wilcoxon rank sum test
[0354] To compare times to outcomes, survival and event-free survival was charted with Kaplan-Meier curves and compared by the log-rank test.
Sample Size
[0355] The power calculation was based on difference in change of CO and conducted as described in Table 3. With a power of 80%, 2-sided alpha of 0.05 and an assumed 10% minimal treatment difference the sample size required 10 patients in each group. Accounting for potential missing data measurements and additional margins, sample size was set to 15 patients in each group.
TABLE-US-00007 TABLE 3 Sample size calculation Assumptions: Standard deviation of difference 0.4 in change of CO from baseline to 120 minutes (L/min) Mean CO (L/min) 4.2 5% increase from 4.2 L/min 0.21 10% increase from 4.2 L/min 0.42 15% increase from 4.2 L/min 0.63 Significance set to 0.05 (2-sided) power: 0.80 including additional 10% for Minimal detectable difference Treated total anticipated (treatment effect) n = n = missing data 5% 31 62 68 10% 10 20 22 15% 6 12 13 Decision: 10% with margins --> 15 30 30
Ethics
[0356] Ghrelin has been used previously in studies in healthy humans and patients. The trial was conducted according to International Conference on Harmonization and Good Clinical Practice guidelines and the Declaration of Helsinki. The trial was approved by the local (Stockholm) ethics committee (number 2008/1:12 and 2008/1695-31). In this physiological study, the ethics committee waived the need for medical products agency (MPA) approval, based on a previous waiver for the same treatment in another study of gastrointestinal effects (MPA waiver number 159:2007/16373; ethics Stockholm number 2007/119-31/1). All patients provided written informed consent.
Methods of the Rodent Studies
Mouse Model of Myocardial Infarction-Induced Heart Failure
[0357] Twelve-week old C57BL6 mice were anesthetized with gas mixture of oxygen and isoflurane (2-3%). 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 and subsequent left coronary ligation; ˜80% survived during the follow-up period (4-6 weeks). SHAM-operated animals underwent the same procedure without coronary artery ligation (SHAM). At study termination, after sedation, mice were euthanized through cervical dislocation.
Mouse Transthoracic Echocardiography
[0358] Heart function was noninvasively monitored in both groups by transthoracic echocardiography using the Philips HDI 5000 imaging system before the termination. Echocardiograms were performed with a 7-15 MHz CL 15-7 scanning head. Heart contractility was measured as % left ventricular fractional shortening using the following formula: LVd-LVs/LVd*100, where LVd and LVs stand for left ventricular diastolic and systolic dimensions respectively.
Mouse Cardiomyocyte Isolation
[0359] After euthanasia, single cardiomyocytes were isolated from the left and right ventricles (mouse hearts are dominated by left ventricles) and atria were excluded, following the protocols developed by the Alliance for Cellular Signalling (AfCS Procedure Protocol ID PP00000 125) as previously described (Pironti et al., 2016).
Cytosolic [Ca2+]I and Cell Shortening in Response to Ghrelin and Placebo
[0360] Mouse cardiomyocytes were incubated with a cell permeable form of fluorescent indicators Fluo-3 AM followed by washing for >5 min as previously described (Andersson et al., 2011). Cardiomyocytes were plated on laminin coated glass bottom dishes. The dishes were placed in a custom built perfusion/stimulation chamber and continuously perfused with O.sub.2/CO.sub.2 (95/5%) bubbled Tyrode solution with the following composition (in mM): NaCl 121, KCl 5.0, CaCl.sub.2 1.8, MgCl.sub.2 0.5, NaH2PO.sub.4 0.4, NaHCO.sub.3 24, EDTA 0.1, glucose 5.5. The cardiomyocytes were stimulated to contract using an electrical field between two platinum electrodes attached to the perfusion/stimulation chamber. Measurements were only performed in cardiomyocytes that contracted upon electrical stimulation and displayed normal morphology (e.g. striated, “brick shaped”). Cells that displayed spontaneous contractions were not measured. Fluorescence was measured using a confocal microscope Bio-Rad MRC 1024 unit attached to a Nikon Diaphot inverted microscope (40-60×oil immersion lenses). Confocal images were analyzed off line using ImageJ (National Institutes of Health; available at http://rsb.info.nih.gov/ij). Line scan confocal images were obtained from paced cardiomyocytes with the line scan running along the long axis of the cell. Changes in the emitted fluorescence, representing changes in free cytoplasmic Ca.sup.2+, were quantified. The amplitude of Ca.sup.2+ transients was measured as the change in the fluo-3 fluorescence signal (F) divided by the fluorescence immediately before a stimulation pulse given under control conditions (FO). Ca.sup.2+ transient decay time constant (Tau) was quantified by fitting the decay to an exponential decay function in the GraphPad Prism software (La Jolla, Ca, USA). Fractional cell shortening was calculated from the line scan images as the fractional change in the cell length at rest and during maximal contraction.
[0361] The myocytes were superfused with physiological buffer (Tyrode solution) or for the pharmacological experiments Tyrode+Ghrelin (100 nM) or D-Lys 3 (D-Lys 3 GHS-R1a antagonist 3 μM). All cells were perfused for 15 min with respective solution before measurements, except in some experiments where D-Lys 3 (3 μM) was introduced in perfusion system 10 minutes before adding Ghrelin. All experiments were performed at room temperature (˜24° C.).
Ghrelin Treatment of Cardiomyocytes
[0362] The myocytes were superfused with physiological buffer (Tyrode solution) with or without Ghrelin (Bachem, Bubendorf, Switzerland) (100 nM). All cardiomyocytes were superfused for 15 min with respective solution before measurements. An antagonist against the ghrelin receptor GHS-r1a (D-Lys 3; 3 μM), was used in some experiments. In those experiments, D-Lys 3 was introduced in superfusion system 10 minutes before adding Ghrelin.
Mouse Cardiomyocyte Protein Immunoblotting
[0363] Isolated cardiomyocytes were treated for 15 min in different conditions (Placebo, Ghrelin, D-Lys 3 GHS-R1a, D-Lys 3 GHS-R1a+Ghrelin). Thereafter cardiomyocytes were pelleted and homogenated. Protein lysates were separated by electrophoresis and transferred onto membranes. Membranes were incubated with primary antibody: rabbit phospho-troponin I (cardiac) (Ser 23/23) antibody (Cell Signaling #4004), mouse troponin 1 (Millipore MAB1691A). Then infrared-labelled secondary antibodies (IRDye 680 and IRDye 800, 1:5000, Licor) were used. Immunoreactive bands were analyzed using the Odyssey Infrared Imaging System. Band densities were quantified with Image J, normalized to GAPDH and final data expressed as fold increase compared to Placebo group.
cAMP Measurement
[0364] The intracellular cAMP concentration was measured using the cAMP direct Immunoassay kit (Abcam ab65355). Briefly, frozen cardiomyocytes pellets from groups described above (Placebo, Ghrelin, D-Lys 3 GHS-R1a+Ghrelin) were homogenized on ice using volume of 0.1 M HCl to obtain a protein concentration of 1 mg/ml. Acetylated supernatants of samples were used for the incubation with antibodies in ELISA plates according to the kit manufacturer. Finally, the optic density absorbance at 450 nm was analyzed using a microplate reader (Biotek, Synergy 2). The concentration of cAMP was normalized, dividing the resulting readout (pmol/ml) by the total protein concentration (mg/ml) for each sample. The experiments were conducted by an operator blinded regarding the treatment of the animals.
Statistics
[0365] Statistical comparison between 2 groups were performed using students t-test (unpaired). For comparison between >2 groups, ANOVA was used. A p<0.05 was used as definition for statistical significance. Average data was presented as mean±standard error of the mean (SEM).
Ethical Approval
[0366] All animal experiments were performed under the ethics approvals N19/15, N273/15.
Results of the Human Trial
Patients
[0367] Thirty-four patients were identified from Karolinska University Hospital heart failure clinic and consented to the trial. On the morning of study, one patient was excluded because unable to lie still due to neuropathy and leg pain, one patient because of creatinine clearance 26 mL/min (<30 mL/min was exclusion criterion), and one patient because of NYHA class II (NYHA III-IV was inclusion criterion) and EF 43% (EF≤40% was inclusion criterion). Thus thirty-one patients were randomized. One patient (placebo) experienced repeated urinary urgency and dizziness early after start of infusion and infusion was interrupted and patient excluded. Thirty patients remained for analysis (15 ghrelin; 15 placebo).
Baseline Characteristics
[0368] Baseline characteristics are shown in Table 4 and were similar between the treatment and placebo groups. Median age was 71 and 70, respectively, and 13% were women in both groups. Baseline echocardiography data are shown in Table 5. Median EF (Simpson) was 30% and 28%, respectively.
TABLE-US-00008 TABLE 4 Baseline clinical characteristics. Continuous variables are presented as medians (IQR) and categorical variables as n and %. p values were assessed by Wilcoxon rank sum test for continuous variables and by Fischer’s exact test for categorical variables. Additional biomarkers are shown in Table 12. Ghrelin Placebo Missing n = 15 n = 15 p Variable n (%) (50%) (50%) value Demographics Age (years) 70 (62-76) 71 (55-75) 0.787 Male sex, n (%) 13 (87) 13 (87) 1.000 BMI (kg/m.sup.2) 29 (25-30) 30 (28-32) 0.097 Vital parameters SBP (mmHg) 105 100 (100- 0.738 (95-120) 120) DBP (mmHg) 70 (65-80) 70 (65-80) 0.655 SpO.sub.2 (%) 97 (95-98) 96 (95-97) 0.421 HF related Duration of HF (years) 8 (2-16) 9 (5-13) 0.967 NYHA class III, n (%) 14 (93) 14 (93) 1.000 NYHA class IV 1 (7) 1 (7%) 1.000 Ischemic cardiomyopathy, n 7 (47) 11 (73) 0.264 (%) Biochemistry P-acyl ghrelin fasting (ng/L) 70 (32-144) 82(30-97) 0.724 (normal fasting 5%-95% range 27-328 ng/L*) Medical history n (%) Previous cardiovascular 11 (73) 11 (73) 1.000 disease Whereof 6 (55) 11 (100) 0.035 Previous MI Previous CABG 3 (27) 7 (63) 0.198 Previous PCI 5 (45) 4 (36) 1.000 Previous Stroke/TIA 2 (18) 2 (18) 1.000 Claudication 3 (27) 2 (18) 1.000 Atrial fibrillation or flutter 12 (80) 10 (67) 0.682 Previous valve disease, at 4 (27) 5 (33) 1.000 least moderate Hypertension 11(73) 8 (53) 0.450 Hyperlipidemia 10(67) 1 (73) 1.000 Pulmonary disease 4 (27) 3 (20) 1.000 Previous or current 5 (33) 2 (13) 0.390 malignancies Inflammatory disease 1 (3) 2 (14) 1 (7) 0.598 (polymyalgia reumatica, rheumatoid arthritis) Smoking ever 10 (67) 11 (73) 1.000 Smoking current 2 (13) 3 (20) 1.000 Diabetes Mellitus 7 (47) 6 (40) 1.000 Treatment, n (%) ACEi/ARB 15 (100) 15 (100) 1.000 Percent dose <50% 2 (13) 2 (13) 1.000 50-99% 3 (20) 3 (20) ≥100% 10 (67) 10 (67) Beta blockers 15 (100) 15 (100) 1.000 <50% 1 (7) 0 0.222 50-99% 5 (33) 2 (13) ≥100% 9 (60) 13 (87) MRA 11 (73) 14 (93) 0.330 Loop diuretics 1 (3) 12 (86) 14 (93) 0.598 Thiazide diuretics 0 0 Levosimendan ever 3 (20) 3 (20) 1.000 Other inotropes ever 0 (7) 1 (7) 1.000 Non-dihydropyridine Calcium 1 (3.3) 0 (0) 1 (7) 0.483 channel blocker Aspirin 2 (6.7) 3 (23) 3 (20) 1.000 Other anti-platelets 1 (7) 1 (7) 1.000 Warfarin 2 (6.7) 10 (77) 11 (73) 1.000 Low molecular heparin 1 (3.3) 0 1 (7) 1.000 Digoxin 1 (7) 3 (20) 0.598 Amidarone 2 (13) 3 (20) 1.000 Statins 9 (60) 10 (66) 1.000 Allopurinol 3 (20) 3 (20) 1.000 Insulin 2 (13) 6 (40) 0.333 Oral glucose lowering 3 (20) 6 (40) 0.427 treatment CRT 8 (53) 10 (67) 0.710 ICD 14 (93) 11 (73) 0.330 Secondary prevention 5 (33) 2 (13) 0.390 Primary prevention 9 (60) 10 (66) 1.000 Tests Previous VO.sub.2 max test, n (%) 8 (57) 9 (60) 1.000 Most recent VO.sub.2 max, 13.4 (11.6, 12.2 (9.1, 0.5625 16.4) 14.1) Previous 6 minutes' walk test, 5 (33) 3 (20) 0.682 n (%) Most recent 6 minutes' walk 426 330 0.1771 test (m) (421-450) (330-355) Symptoms the week prior to inclusion, n (%) Paroxysmal nocturnal 0 2 (13) 1 (7) 1.000 dyspnea Orthopnea 0 4 (27) 4 (27) 1.000 Able to walk one block 0 11 (73) 9 (60) 0.700 Dyspnea at rest 0 0 (0) 3 (20) 0.224 Unspecific chest pain, any 0 1 (7) 3 (20) 0.598 cause ECG, n (%) Sinus rhythm 0 5 (33) 7 (47) 0.710 Atrial fibrillation/flutter 0 5 (33) 7 (47) 0.710 Atrial pace 0 5 (33) 1 (7) 0.169 Ventricular pace 0 7 (47) 10 (67) 0.462 Abbreviations: BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; SpO2, peripheral saturation by pulse oximetry, HF, heart failure; NYHA class, New York Heart Association class; INR, international normalized ratio; NT-proBNP, N-terminal pro brain natriuretic peptide; hsCRP, high sensitive C reactive protein; MI, myocardial infarction; AP, angina pectoris; CABG, coronary artery bypass grafting; PCI, percutaneous coronary intervention; TIA, transient ischemic attack, CRT, cardiac resynchronization therapy; ICD, implantable cardioverter defibrillator; MRA, mineralocorticoid receptor antagonist; ACEi, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker. *D Webb, data on file from 41 healthy non-obese adults
TABLE-US-00009 TABLE 12 Biomarkers at baseline and 2-5 days follow up Ghrelin Placebo P 2-5 2-5 value days days inter- Variable Baseline FU Baseline FU action NT- 2080 2450* 2380 2020 0.01 proBNP [1080-3980] [942-4920] [787-4060] [762-2870] [ng/L] Troponin 21 ± 10 31 ± 38 22 ± 13 24 ± 15 0.36 T [ng/L] Creatinine 112 ± 34 123 ± 35* 118 ± 33 124 ± 37 0.28 [ukat/L] Cystatin 1.45 ± 10.44 1.59 ± 0.52* 1.45 ± 0.42 1.62 ± 0.50* 0.57 C [mg/L] hsCRP 2.7 3.0 2.4 3.0 0.63 [mg/L] [0.9-7.7] [1.3-8.0] [1.1-7.8] [1.0-5.6] Insulin 36 30 39 28 0.70 (non- [20-55] [12-59] [27-49] [20-61] fasting) [mlE/L] ASAT 0.41 0.42 0.37 0.40 0.41 [ukat/L] [0.33-0.47] [0.33-0.50] [0.27-0.45] [0.28-0.47] ALAT 0.31 0.31 0.35 0.35 0.70 [ukat/L] [0.24-0.38] [0.26-0.41] [0.25-0.48] [0.23-0.43] GT 0.77 0.83 0.97 1.00 0.60 [ukat/L] [0.65-1.40] [0.66-1.30] [0.47-1.50] [0.47-1.60] Tri- 1.4 1.6 1.6 1.6 0.94 glycerides [0.9-1.48] [1.1-2.7]* [0.9-1.9] [1.3-2.8]* [mmol/L] INR 2.2 2.6 2.6 2.5 0.02 among [2.0-2.8] [2.2-2.9] [2.4-2.8] [2.3-2.5] warfarin treated INR 1.1 1.0 1.1 1.0 0.19 among [1.0-2.3] [1.0-2.7] [1.0-1.2] [0.9-1.1] non- warfarin treated Potassium 4.1 ± 10.6 4.3 ± 0.4 4.1 ± 0.4 4.2 ± 0.4 0.88 [mmol/L] Sodium 139 ± 13 138 ± 12 138 ± 13 138 ± 12 0.16 [mmol/L] Haemog- 13.5 ± 114 13.5 ± 17 12.9 ± 17 13.1 ± 17 0.20 lobin [g/dL] Haemato- 40 ± 4 41 ± 5 38 ± 4 39 ± 4 0.96 crit [%] White 7.4 ± 1.9 7.4 ± 2.0 7.0 ± 1.7 7.3 ± 1.3 0.23 blood cell count (109/L) * = p < 0.05 vs. baseline within the same group (treatment or placebo)
Patient Outcomes
Plasma Acyl Ghrelin Pharmacokinetics
[0369] Plasma acyl ghrelin concentrations at baseline and during and after ghrelin infusion are shown in Table 6 and
TABLE-US-00010 TABLE 6 Ghrelin pharmacokinetics. Measured plasma acyl ghrelin in placebo and treatment group (median, interquartile range). The lower limit of detection was as 3.2 ng/L P for trend 30 min post infusion to 2-5 days post infusion, within ghrelin group p < 0.001 and within placebo group p = 0.0254. Ghrelin, acyl Placebo, acyl P Time ghrelin (ng/L) ghrelin (ng/L) value Fasting 70(32-144) 82(30-97) 0.724 Prior to infusion 76(42-131) 60(40-78) 0.361 Infusion 30 min 6114(5102-8267) 62(35-90) <0.001 Infusion 60 min 6841(4755-11 392) 64(32-85) <0.001 Infusion 120 min 6536(4835-10 078) 59(37-73) <0.001 30 min post infusion 503(372-833) 58(37-89) <0.001 2-5 days post infusion 139 (117-161) 55(31-69) 0.001 P trend 0 to 120 min <0.0001 0.7893 within groups P trend 0 to 120 min <0.0001 between groups
Primary Efficacy Outcome: Cardiac Output
[0370] Tables 7 and 8 and
TABLE-US-00011 TABLE 7 Acute hemodynamic effects of ghrelin vs. placebo infusion Ghrelin Placebo Infusion + 30 min 30 min after P value Infusion + 60 120 after stop Infusion + Infusion + stop inter- Variable Baseline min min Infusion Baseline 60 min 120 min infusion action CO [L/min] 4.08 ± 1.15 4.64 ± 1.31* 5.23 ± 1.98* 4.94 ± 1.70 4.26 ± 1.23 3.99 ± 1.16 4.11 ± 1.99 3.70 ± 0.99 <0.001 SV [ml] 59.10 ± 17.82 69.64 ± 22.75* 79.83 ± 30.30* 74.31 ± 26.09 62.0 ± 15.7 58.9 ± 17.7 60.7 ± 15.1 55.1 ± 15.9 <0.001 HR [bpm] 70.7 ± 11.3 68.5 ± 11.2 67.1 ± 11.1 68.2 ± 11.4 68.7 ± 7.6 68.6 ± 8.1 67.6 ± 9.5 68.1 ± 8.6 0.15 VO.sub.2 [L/min] 0.20 ± 0.07 0.19 ± 0.07 0.19 ± 0.08 0.19 ± 0.08 0.20 ± 0.08 0.20 ± 0.09 0.19 ± 0.08 0.18 ± 0.08 0.88 A-V O.sub.2 diff [%] 27.6 ± 14.3 23.6 ± 12.9* 20.9 ± 8.5* 21.6 ± 12.1 26.4 ± 9.0 28.5 ± 11.3 26.6 ± 10.7 29.0 ± 15.1 0.0049 SpO.sub.2 [%] 95.2 ± 3.4 93.5 ± 3.5* 93.4 ± 3.6* 94.7 ± 3.9 95.7 ± 1.9 95.2 ± 2.0 95.7 ± 1.8 95.9 ± 2.3 0.0054 Shunt [%] 9.9 ± 10.6 16.8 ± 11.8* 18.0 ± 12.3* 12.6 ± 14.3 9.5 ± 6.7 11.7 ± 10.1 10.5 ± 8.6 10.3 ± 10.7 0.0083 SvO.sub.2 [%] 67.9 ± 14.4 70.0 ± 13.8 71.7 ± 10.1 73.3 ± 12.8 69.3 ± 8.6 66.8 ± 10.5 69.1 ± 9.9 67.1 ± 14.6 0.094 PBF [L/min] 3.61 ± 0.70 3.76 ± 0.76 4.08 ± 0.90* 4.14 ± 0.97 3.82 ± 1.06 3.52 ± 1.08* 3.66 ± 1.12 3.30 ± 1.02 0.008 dBP [mmHg] 58.6 ± 8.4 56.5 ± 8.7 56.1 ± 10.3 58.9 ± 7.5 63.5 ± 10.1 66.5 ± 8.0 63.4 ± 8.2 68.4 ± 10.1 0.11 sBP [mmHg] 104.7 ± 18.4 99.0 ± 17.6 99.0 ± 21.1 105.7 ± 20.5 120.2 ± 24.4 118.2 ± 21.3 115.5 ± 22.3 123.6 ± 25.2* 0.83 mBP [mmHg] 74.9 ± 11.3 71.4 ± 10.2 70.9 ± 13.1 75.5 ± 12.0 82.2 ± 14.1 84.1 ± 11.9 81.1 ± 12.1 87.0 ± 14.5* 0.23 dP/dt [mmHg/s] 742 ± 267 535 ± 213 531 ± 191 602 ± 252 875 ± 305 675 ± 256 699 ± 265 776 ± 314 0.81 eSVR 1567 ± 468 1311 ± 365* 1200 ± 387* 1354 ± 480 1656 ± 512 1820 ± 574 1693 ± 484 2013 ± 658 0.0004 [dyn*s/ cm-1]] * = p < 0.05 vs. baseline within the same group (treatment or placebo) † = p < 0.05 vs. infusion 60 min within the same group (treatment or placebo) ‡ = 0.05 < p > 0.10 vs. baseline within the same group (treatment or placebo) Measured data: VO2, SpO2, HR, PBF, dBP, sBP Calculated data: CO, SV, A-V O2 diff, Shunt, SvO2, mBP, eSVR
TABLE-US-00012 TABLE 8 Hemodynamics at baseline and 2-5 day follow-up visit P value Ghrelin Placebo inter- Variable Baseline Follow-up Baseline Follow-up action CO [L/min ] 4.08 ± 1.15 4.21 ± 1.27 4.26 ± 1.23 3.98 ± 1.05.sup.† 0.017 SV [ml] 59.9 ± 19.0 59.9 ± 18.5 61.9 ± 15.2 57.4 ± 14.3.sup.† 0.16 HR [bpm ] 69.6 ± 9.1 71.6 ± 10.8 68.8 ± 7.6 69.8 ± 9.8 0.53 VO.sub.2 [L/min ] 0.2 ± 0.07 0.22 ± 0.06 0.20 ± 0.09 0.23 ± 0.09 0.97 SpO.sub.2 [% ] 95.2 ± 3.4 94.0 ± 3.9+ 95.4 ± 1.7 95.6 ± 1.3 0.028 SvO.sub.2 [% ] 67.7 ± 14.3 66.4 ± 18.4.sup.† 69.9 ± 8.5 64.9 ± 8.6.sup.† 0.82 A-V O.sub.2-diff [%] 24.4 ± 7.6 27.4 ± 10.0.sup.‡ 25.6 ± 8.7 30.7 ± 9.0.sup.† 0.41 Shunt [% ] 9.9 ± 10.6 15.7 ± 16.7 10.2 ± 6.4 8.6 ± 5.1 0.09 PBF [L/min ] 3.61 ± 0.70 3.58 ± 0.74 3.88 ± 1.08 3.64 ± 0.87.sup.‡ 0.30 .sup.† = p < 0.05 vs. baseline in the same group (GHSRA or placebo) .sup.‡ = p = 0.05-0.10 vs. baseline in the same group (GHSRA or placebo) The ghrelin molecule is “GHSRA”.
Secondary Efficacy and Safety Outcomes: Hemodynamics
[0371] Tables 7 and 8 and
[0372] Tables 7 and 8 and
[0373] Tables 7 and 8 and
[0374] Tables 7 and 8 show additional hemodynamic data. There was no change in oxygen consumption (VO.sub.2), suggesting that neither ghrelin nor placebo affected metabolism (oxygen demand). Pulmonary blood flow increased in the ghrelin groups, consistent with the increase in CO.
Secondary Outcomes: Echocardiography
[0375] Tables 9 and 10 and
Biomarkers
[0376] Table 11 shows biomarkers before, during infusion, and 30 minutes after infusion. There was no interaction with treatment for any biomarker. There was no interaction with or change in troponin T. NT-proBNP increased in both groups, which is likely due to prolonged supine state and not taking diuretics during the infusion. Cystatin C increased in both groups. Table 12 shows biomarkers at 2-5 days after infusion. NT-proBNP remained elevated in the ghrelin group. This may be due to a stimulating effect of ghrelin leading to increased activity after infusion or different times since infusion. With multiple testing, it may also be due to chance.
Safety Outcomes During and Immediately after Infusion and 2-5 Days after Infusion
[0377] There was no effect of ghrelin or placebo on heart rate (
[0378] Among symptoms (headache, dizziness, dyspnea, central chest pain, flush, sleepiness, upset stomach and other symptoms) assessed as yes or no, flush at 60 min was more frequent in the intervention group compared to placebo (7 vs. 0; p=0.006) There was a nominal but not significant increase in QTc (Table 13 and
TABLE-US-00013 TABLE 11 Acute effects of ghrelin vs. placebo on biomarkers Ghrelin Placebo Infusion Infusion Infusion Infusion Infusion Infusion 30′ p- + + + 30′ after + + + after inter- Variable Baseline 30′ 60′ 120′ infusion Baseline 30′ 60′ 120′ infusion action NT- 2080 2220 2340* 2630* 2820 2380 2410 2490* 2750* 2820 0.63 proBNP [1080- [1080- [1100- [1220- [1280- [787- [754- [918- [860- [968- [ng/L] 3980] 4030] 4580] 4840] 5170] 4060] 4000] 4770] 4900] 4690] Troponin T 21 ± 9 21 ± 9 22 ± 10 21 ± 10 21 ± 10 22 ± 13 21 ± 13 22 ± 13 22 ± 13 23 ± 13 0.67 [ng/L] Creatinine 112 ± 34 110 ± 31 112 ± 30 114 ± 29 113 ± 27 118 ± 33 114 ± 117 ± 32 118 ± 32 119 ± 32 0.58 [ukat/L] 32* Cystatin C 1.45 ± 0.44 1.46 ± 0.44 1.55 ± 0.47* 1.57 ± 0.43* 1.54 ± 0.41 1.45 ± 0.42 1.42 ± 0.40 1.49 ± 0.37 1.51 ± 0.38* 1.57 ± 0.40 0.48 [mg/L] hsCRP 2.7 3.0 3.0 3.3 3.5 2.4 2.3 2.4 2.3 2.4 0.75 [mg/L] [0.9-7.7] [1.1-7.6] [1.1-7.6] [1.1-7.5] [1.1-7.8] [1.1-7.8] [1.0-7.6] [0.9-7.5] [1.1-7.8] [1.0-8.1] ASAT 0.41 0.40 0.44 0.41 0.43 0.37 0.33 0.40 0.37 0.34 0.97 [ukat/L] [0.33-0.47] [0.34-0.43] [0.36-0.47] [0.33-0.46] [0.36-0.49] [0.27-0.45] [0.28-0.43] [0.30-0.44] [0.24-0.45] [0.30-0.50] ALAT 0.31 0.31 0.31 0.31 0.32 0.35 0.34 0.35 0.34 0.33 0.58 [ukat/L] [0.24-0.38] [0.23-0.39] [0.31-0.40] [0.21-0.39] [0.23-0.41] [0.25-0.48] [0.23-0.48] [0.23-0.49] [0.23-0.48] [0.22-0.47] GT 0.77 0.78 0.79 0.80 0.82 0.97 0.93 0.94 0.95 0.98 0.51 [ukat/L] [0.65-1.40] [0.65-1.40] [0.62-1.40] [0.62-1.30] [0.62-1.40] [0.47-1.50] [0.47-1.50] [0.46-1.50] [0.48-1.50] [0.48-1.50] * = p < 0.05 vs. baseline within the same group (treatment or placebo)
TABLE-US-00014 TABLE 13 Effects of ghrelin and placebo on QTc interval During infusion After infusion 60 minutes 120 minutes Prior to discharge 2-5 days after Median Placebo −3 (−8 to +14) −4 (−10 to +11) +4 (−3 to +14) +2 (−16 to +20) (interquartile Ghrelin +4 (−7 to +21) +8 (−4 to +26) +18 (−10 to +30) −4 (−34 to +4) range) changes in P placebo vs 0.418 0.191 0.295 0.135 QTc (ms) from ghrelin baseline Maximal QTc Placebo +60 +80 +96 +103 prolongation (ms) Ghrelin +36 +45 +47 +47 from baseline