In vitro method and use of pro-adrenomedulin or fragments thereof to assess congestion in an individual with acute heart failure.

Abstract

The present invention relates to a method for a) diagnosing congestion or assessing or monitoring the extent of congestion in an individual, or b) predicting or determining or monitoring the need for therapy or intervention, or predicting or determining or monitoring the success of a congestion therapy or intervention, or guiding a therapy or intervention in an individual, or c) predicting decongestion or residual congestion after congestion therapy or intervention in an individual, or d) assessing decongestion or residual congestion after congestion therapy or intervention in an individual, or e) evaluating the decision regarding hospital discharge of an individual.wherein said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF or wherein said individual has chronic heart failure with signs of worsening / symptoms of chronic heart failure and wherein pro-adrenomedullin or its fragments of at least 5 amino acids is used as an early surrogate marker for congestion.

Description

1 / 98 Descriptive Report of the Invention Patent for an IN VITRO METHOD AND USE OF PRO-ADRENOMEDULIN OR FRAGMENTS THEREOF TO EVALUATE CONGESTION IN AN INDIVIDUAL WITH ACUTE HEART FAILURE.

[001] The subject matter of the present invention is a method for: a) to diagnose congestion or to assess or monitor the extent of congestion in an individual or, b) to predict or determine or monitor the need for congestion therapy or intervention, or to predict or determine or monitor the success of a congestion therapy or intervention, or to guide a therapy or intervention in an individual, or c) to predict residual decongestion or congestion after congestion therapy or intervention in an individual or, d) to assess residual decongestion or congestion after congestion therapy or intervention in an individual; or (e) assess the decision regarding hospital discharge of an individual,

[002] wherein said individual is an individual who has acute heart failure that is newly onset AHF or acute HF or acute decompensated chronic HF or wherein said individual is an individual who has chronic heart failure with signs of worsening / symptoms of chronic heart failure and wherein the measurement of Pro-Adrenomedullin or its fragments of at least 5 amino acids is used as an early surrogate marker for congestion.

[003] Heart failure (HF) is a heart condition that occurs when there is a problem with the structure or function of the heart. Petition 870230018821, dated 06 / 03 / 2023, page 9 / 138 2 / 98 impairs the ability of the heart to provide sufficient blood flow to meet the body's needs. It can cause a wide variety of symptoms, particularly shortness of breath (SOB) at rest or during exercise, signs of fluid retention such as pulmonary congestion or ankle swelling, and objective evidence of an abnormality in the structure or function of the heart at rest.

[004] Heart failure is a clinical syndrome characterized by a set of symptoms and signs caused by cardiac dysfunction. It is one of the leading causes of morbidity and mortality in developed countries, with a prevalence of 1 to 2%. Heart failure can be grouped into chronic FH and acute FH. Patients with chronic FH can be grouped into stable chronic FH, signs and symptoms of chronic FH, and acute decompensation of chronic FH. Acute heart failure (AHF) is defined as a rapid onset of signs and symptoms of heart failure, resulting in the need for urgent therapy or hospitalization. AHF can present as new acute FH (new onset of AHF in a patient without previous cardiac dysfunction) or acute decompensation of chronic FH. AHF is the leading cause of hospitalization in adults older than 65 years of age.Despite notable improvements in the prognosis of patients with chronic heart failure, mainly related to therapeutic advances in recent decades, both short-term and long-term outcomes remain very poor once patients are hospitalized for decompensated heart failure. Nearly 25% of patients hospitalized for AHF require readmission within 30 days of hospital discharge, while <50% survive beyond 5 years after hospitalization. In addition to the significantly reduced survival and quality of life of affected patients, there is also the financial burden. Petition 870230018821, dated 06 / 03 / 2023, page 10 / 138 3 / 98 of the AHF's assessment of healthcare systems is enormous. The total cost of treating heart failure was estimated to be $31 billion in the US in 2012 alone, and the majority of this cost is associated with hospital treatment. This cost is projected to increase to an unprecedented $70 billion by 2030 due to aging populations.

[005] Heart failure encompasses a wide range of patients, from those with normal left ventricular ejection fraction (LVEF), typically considered to be > 50%, also known as HF with preserved EF (HFpEF), to those with reduced LVEF, typically considered to be < 40%, also known as HF with reduced EF (HFrEF). Patients with LVEF in the 40-49% range represent a 'grey area', which is defined as HF with semi-amplitude EF (HFmrEF) (Ponikowski et al. 2016. European Heart Journal 18(8): 891-975).

[006] The primary goal of AHF treatment in the hospital setting is decongestion (removal of excess intra- and extracellular fluid, simply put) and relief of the symptoms and signs of congestion. Diuretics remain the primary decongestive therapy in AHF, and almost all hospitalized patients receive this class of drugs. Other classes of medications that increase cardiac output and reduce load pressure, such as inotropes and vasodilators, are administered in selected groups of patients. Ultrafiltration may also be considered in some patients, particularly those who do not respond adequately to diuretic therapy.

[007] Although patients (generally) respond well to diuretic therapy, a significant proportion of patients are discharged from the hospital without achieving an adequate level of decongestion and euvolemic state (i.e., residual congestion). This is mainly Petition 870230018821, dated 06 / 03 / 2023, page 11 / 138 4 / 98 related to the fact that current approaches to the clinical assessment of congestion are inadequate. There is consistent evidence that the presence of residual congestion at hospital discharge is associated with worse post-discharge outcomes, particularly hospital readmissions. Therefore, there is a significant unmet need for a more accurate and reliable surrogate for congestion that can facilitate aiming for and optimal decision-making regarding the adequacy of the level of decongestion achieved and the appropriate timing of hospital discharge.

[008] The adrenomedullin (ADM) peptide was first described in Kitamura et al. (Kitamura et al. 1993. Biochemical and Biophysical Research Communications 192 (2): 553-560) as a novel hypotensive peptide comprising 52 amino acids, which were isolated from a human pheochromocytoma. In the same year, the cDNA encoding for a precursor peptide comprising 185 amino acids and the complete amino acid sequence of this precursor peptide were also described. The precursor peptide, which comprises, inter alia, a 21-amino acid signal sequence at the N-terminus, is referred to as preproadrenomedullin (pre-proADM). Pre-proADM comprises 185 amino acids. Mature ADM-NH2 is presented in SEQ ID NO:. 4 and ADM-Gly is presented in SEQ No.: 5.

[009] Mature adrenomedullin peptide is an amide-converted peptide (ADM-NH2) comprising 52 amino acids (SEQ ID NO: 4) and comprising amino acids 95 to 146 of preproADM, from which it is formed by proteolytic cleavage. To date, substantially only a few fragments of the peptide fragments formed in the cleavage of preproADM have been more precisely characterized, in particular the physiologically active peptides adrenomedullin (ADM) and PAMP, a peptide comprising 20 amino acids (22 to 41) that follows the 21 amino acids of the signal peptide in preproADM. Furthermore, for Petition 870230018821, dated 06 / 03 / 2023, page 12 / 138 5 / 98 Both ADM and PAMP, the physiologically active sub-fragments, were verified and investigated in greater detail. The finding and characterization of ADM in intensive research activity triggered in 1993 and a flurry of publications, the results of which have recently been summarized in several analytical articles, in the context of the present description, reference being made in particular to the articles Takahashi 2001. Peptides 22:1691; Eto et al. 2001. Peptides 22: 1693-1711 and Hinson et al. 2000 Endocrine Reviews 21(2):138-167.

[0010] In scientific investigations to date, it has been observed, among other things, that ADM can be considered a polyfunctional regulatory peptide. It is released into the circulation partially in an inactive form extended by glycine (Kitamura et al. 1998. Biochem. Biophys. Res. Commun. 244(2): 551-555). There is also a binding protein (Pio et al. 2001. The Journal of Biological Chemistry 276(15): 12292-12300), which is specific for ADM and probably also modulates the effect of ADM.

[0011] These physiological effects of ADM, as well as PAMP, which are of primary importance in investigations to date, were the effects that influenced blood pressure. Thus, ADM is an effective vasodilator.

[0012] Furthermore, it has been found that the aforementioned additional physiologically active peptide PAMP formed from pre-proADM also exhibits a hypotensive effect, even though it appears to have a different mechanism of action from that of ADM (Eto et al. 2001. Peptides 22: 1693-1711; Hinson et al. 2000 Endocrine Reviews 21(2):138-167; Kuwasako et al. 1997. FEBS Lett 414(1): 105-110; Kuwasaki et al. 1999. Ann. Clin. Biochem. 36: 622-628; Tsuruda et al. 2001. Life Sci. 69(2): 239-245; Kangawa et al. EP 0 622 458).

[0013] In addition, it was found that ADM concentrations, Petition 870230018821, dated 06 / 03 / 2023, page 13 / 138 6 / 98, which can be measured in circulation and other biological fluids, are found in various pathological states to be significantly higher than the concentrations found in healthy control individuals. Thus, the ADM level in patients with congestive heart failure, myocardial infarction, renal disease, hypertensive disorders, diabetes mellitus, in the acute phase of shock, and in sepsis and septic shock is significantly increased, despite the different extents. PAMP concentrations are also increased in some of the so-called pathological states, but plasma levels are reduced relative to ADM (Eto et al. 2001. Peptides 22: 1693-1711).

[0014] Furthermore, unusually high concentrations of ADM should be observed in sepsis or septic shock (Eto et al. 2001. Peptides 22: 1693-1711; Hirata et al. 1996. Journal of Clinical Endocrinology and Metabolism 81(4): 1449-1453; Ehlenz et al.1997. Exp Clin Endocrinol Diabetes 105: 156-162; Tomoda et al. 2001. Peptides 22: 1783-1794; Ueda et al. 1999 Am. J. Respir. Crit. Care Med.160: 132-136; Wang et al. 2001. Peptides 22: 1835-1840). The findings are related to typical hemodynamic changes that are known as typical phenomena of the course of a disease in patients with sepsis and other severe syndromes, such as, for example, SIRS. Adrenomedulin plays important roles during the development of sepsis (Wang, Shock 1998, 10(5):383-384; Wang et al. 1998. Archives of surgery 133(12): 1298-1304) and in numerous acute and chronic diseases (Parlapiano et al. 1999). European Review for Medical and Pharmacological Sciences 3:53-61; Hinson et al. 2000 Endocrine Reviews 21(2):138-167).

[0015] Several methods have been described for measuring circulating ADM levels: ADM directly or indirectly through the determination of a more stable fragment of its cognate precursor peptide. Recently, a method describing a Petition 870230018821, dated 06 / 03 / 2023, p. 14 / 138 7 / 98 assay to measure circulating mature ROM (Marino et al. 2014. Crit Care 18: R34).

[0016] Other methods for quantifying ADM precursor-derived fragments have been described, for example, the measurement of MRproADM (Morgenthaler et al. 2005. Clin Chem 51(10):1823-9), PAMP (Washimine et al. 1994. Biochem Biophys Res Commun 202(2):1081-7) and CT-proADM (EP 2 111 552). A commercial homogeneous time-deciduous fluoroimmunoassay for the measurement of MR-proADM in plasma in a fully automated system is available (BRAHMS MRproADM KRYPTOR; BRAHMS GmbH, Hennigsdorf, Germany) (Caruhel et al. 2009. Clin Biochem 42(7-8):725-8). Since these peptides are generated in a stoichiometric ratio from the same precursor, their plasma levels are correlated to some extent.

[0017] Plasma ADM concentrations are elevated in patients with heart failure and correlate with disease severity (Hirayama et al. 1999. J Endocrinol 160:297-303; Yu et al. 2001. Heart 86: 155-160). Elevated plasma ADM is an independent negative prognostic indicator in these individuals (Poyner et al. 2002. Pharmacol Rev 54: 233-246).

[0018] The role of MR-proADM in heart failure has been explored in several studies. In the BACH study (Maisel et al. 2010. J. Am. Coll. Cardiol. 55: 2062-2076), MR-proADM was a powerful prognostic indicator for death at 90 days, adding prognostic value beyond natriuretic peptides. Subsequent data from the PRIDE study (Shah et al. 2012. Eur. Heart J. 33: 2197-2205) solidified a potential prognostic role for MR-proADM; among patients, MR-proADM had the best area under the curve (AUC) with respect to 1-year mortality. Similarly, MR-proADM levels in patients with chronic heart failure (CHF) were strongly correlated with disease severity and elevated levels of Petition 870230018821, dated 06 / 03 / 2023, page 15 / 138 8 / 98 peptides were strongly associated with an increased risk of death at 12 months of follow-up (van Haehling et al. 2010. European Journal of Heart Failure 12: 484-491; Adlbrecht et al. 2009. European Journal of Heart Failure 11: 361-366).

[0019] MR-proADM was investigated during treatment in patients with acute decompensated heart failure (Boyer et al. 2012. Congest Heart Fail 18 (2): 91-97): patients whose MR-proADM levels tended to increase during acute therapy had findings associated with persistent congestion. In the 24-hour period after therapy, patients with elevated MR-proADM had increased peripheral edema. Kaiser et al. measured MR-proADM in patients with univentricular hearts (Kaiser et al. 2014. Europ J Heart Failure 16: 1082-1088). Levels in patients with a faulty Fontan circuit (who present with ascites and peripheral edema) were significantly higher when compared to patients without Fontan failure. Furthermore, Elsenhut speculated whether treatments that lead to a reduction in adrenomedullin levels could reduce the severity and extent of alveolar edema in pneumonia and septicemia (Eisenhut 2006. Crit Care 10: 418).

[0020] Surprisingly, according to the present invention, it has been found that Pro-Adrenomedullin or its fragments are an early and accurate substitute for congestion in the setting of heart failure or acute heart failure, in particular, in an individual who has acute heart failure, which is newly onset AHF or acute decompensated HF or acute decompensated chronic HF or in an individual having chronic heart failure with signs of worsening / symptoms of chronic heart failure.

[0021] The subject matter of the present invention is a method for: a) to diagnose congestion or to assess or monitor the extent of congestion in an individual or, Petition 870230018821, dated 06 / 03 / 2023, page 16 / 138 9 / 98 b) to predict or determine or monitor the need for congestion therapy or intervention, or to predict or determine or monitor the success of a congestion therapy or intervention, or to guide a therapy or intervention in an individual, or c) predicting decongestion or residual congestion after congestion therapy or intervention in a),

[0022] wherein said individual is an individual who has acute heart failure that is newly onset AHF or decompensated acute HF or decompensated chronic HF or wherein said individual is an individual who has chronic heart failure with signs of worsening / symptoms of chronic heart failure and wherein Pro-Adrenomedullin or its fragments of at least 5 amino acids is used as an early surrogate marker for congestion comprising:

[0023] · To determine the level of Pro-Adrenocortin or its fragments of at least 5 amino acids in a body fluid obtained from said individual; and a) correlation of said level of Pro-Adrenomedullin or its fragments with the extent of congestion in said individual or diagnosis of congestion, wherein a level elevated above a certain limit is indicative of congestion or the extent of congestion or, b) correlation of said level of Pro-Adrenomedullin or its fragments with the need for or success of a congestion therapy or intervention in said individual, wherein a level below a certain threshold is predictive of successful congestion therapy or intervention, and wherein a level above a certain threshold is indicative of the need for congestion therapy or intervention, or c) correlation of said level of Pro-Adrenomedulina or its Petition 870230018821, dated 06 / 03 / 2023, page 17 / 138 10 / 98 fragments with a prediction of residual decongestion or congestion after congestion therapy or intervention, where a high level above a certain threshold predicts residual congestion after congestion therapy or intervention, while a level below a certain threshold predicts decongestion after congestion therapy or intervention, or d) correlation of said level of Pro-Adrenomedullin or its fragments with decongestion or residual congestion after congestion therapy or intervention, where a level elevated above a certain limit indicates residual congestion after congestion therapy or intervention, while a level below a certain limit indicates decongestion after congestion therapy or intervention, or (e) correlation of said level of Pro-Adrenomedullin or its fragments with the assessment of the decision regarding hospital discharge, where a level elevated above a certain limit means that the individual should not be discharged and where a level below a certain limit means that the individual can be discharged,

[0024] wherein said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRproADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CTproADM according to SEQ ID NO: 6.

[0025] The terms congestion severity, congestion extent, congestion measure, congestion degree, and similar terms are used synonymously throughout this application. Petition 870230018821, dated 06 / 03 / 2023, page 18 / 138 11 / 98

[0026] Mature ADM, bio-ADM and ADM-NH2 are used synonymously throughout this application and is a molecule according to SEQ ID NO: 4.

[0027] Therapy or intervention for congestion in an individual having acute heart failure and / or an individual having heart failure who presents with signs of worsening and / or an individual with symptoms of heart failure or acute heart failure, may be selected from the group comprising the administration of diuretics, administration of inotropes, administration of vasodilators, ultrafiltration, in particular, diuretics.

[0028] Pro-Adrenomedullin or its fragments are an early, quantitative, and accurate surrogate for congestion in the setting of acute heart failure and heart failure, in particular in an individual having acute heart failure and / or an individual having heart failure who presents with signs of worsening and / or an individual with symptoms of heart failure or acute heart failure. Early and accurate surrogate for congestion in the setting of acute heart failure or heart failure means that its concentration and / or level of immune reactivity reflects the extent of congestion, in particular, the actual extent of congestion.

[0029] For the purposes of this descriptive report, the individual is an individual who is experiencing acute heart failure and / or an individual who is experiencing heart failure who presents with signs of worsening and / or an individual with symptoms of heart failure or acute heart failure.In one specific aspect of the invention, said individual has acute heart failure, which is newly onset AHF or acute decompensated HF. In another particular aspect of the invention, said individual has acute decompensated chronic HF or signs of worsening / symptoms of chronic heart failure. In one particular aspect of the invention, said individual has heart failure. Petition 870230018821, dated 06 / 03 / 2023, page 19 / 138 12 / 98 acute cardiac arrest, particularly new-onset AHF.

[0030] The term acute is used to signify rapid onset and to describe exacerbated or decompensated heart failure, with reference to episodes in which a patient can be characterized as having a change in the signs and symptoms of heart failure that result in a need for urgent therapy or hospitalization.

[0031] The term chronic refers to long duration. Chronic heart failure is a long-term condition, usually kept stable by treating the symptoms (stable chronic FH).

[0032] Stable chronic HF is characterized by: (i) presence of a structural or functional heart defect that impairs its ability to provide sufficient blood flow to meet the body's needs, (ii) absence of volume overload (manifested by pulmonary and / or systemic congestion) and / or profound depression of cardiac output (manifested by hypotension, renal failure and / or shock syndrome), and the patient is considered not to require urgent therapy or therapeutic adjustment and does not require hospitalization. Chronic HF with worsening signs and symptoms is characterized by: (i) presence of a structural or functional heart defect that impairs its ability to supply sufficient blood flow to meet the body's needs, (ii) volume overload (manifested by pulmonary and / or systemic congestion) and / or profound depression of Petition 870230018821, dated 06 / 03 / 2023, page 20 / 138 13 / 98 cardiac output (manifested by hypotension, renal failure and / or shock syndrome) and it is considered that the patient does not require urgent therapy and does not require hospitalization, but needs therapeutic adjustment.

[0033] Chronic heart failure can also decompensate (referred to as acute decompensated heart failure or acute chronic decompensated heart failure), which is most commonly the result of an intercurrent illness (such as pneumonia), myocardial infarction, arrhythmias, uncontrolled hypertension, or a patient's failure to maintain fluid restriction, diet, or medication. After treatment, patients with acute decompensated chronic FH can return to a stable chronic compensated state (stable chronic FH).

[0034] New-onset acute FH and acute decompensated chronic FH are characterized by: (i) presence of a structural or functional heart defect that impairs its ability to provide sufficient blood flow to meet the body's needs, (ii) volume overload (manifested by pulmonary and / or systemic congestion) and / or profound depression of cardiac output (manifested by hypotension, renal failure and / or shock syndrome) and the patient is considered not to require urgent therapy or therapeutic adjustment and does not require hospitalization. Acute FH Chronic FH New-onset FH Acute decompensated FH = Acute decompensated chronic FH Signs / symptoms of worsening chronic FH Stable chronic FH Petition 870230018821, dated 06 / 03 / 2023, page 21 / 138 14 / 98

[0035] The above definitions of acute heart failure in which new-onset AHF or acute decompensated HF or acute decompensated chronic HF or signs of worsening / symptoms of chronic heart failure are aligned with Voors et al., European Journal of Heart Failure (2016), 18, 716 - 726.

[0036] Congestion in FH is defined as high left ventricular diastolic pressure associated with signs and symptoms of FH, such as dyspnea, wheezing, and / or edema. These congestion-related signs and symptoms are the main reasons for FH-related hospitalizations.

[0037] Although relieving congestion (and associated signs / symptoms) and achieving euvolemic status remain the primary goal of hospital AHF therapy, there is no standard algorithm or clinical tool for assessing congestion. Current practices regarding the clinical assessment of congestion revolve around signs and symptoms. Physical examination findings such as elevated jugular venous pressure (JVP), peripheral edema, orthopnea, S3 heart sound, and hepatomegaly, or chest X-ray findings such as cardiomegaly and interstitial / alveolar edema are used as surrogate markers for congestion. It should be noted that, apart from a carefully performed JVP assessment, the predictive value of these parameters for detecting congestion is modest to moderate. There is a significant unmet need for a reliable and accurate surrogate marker for congestion.There is a significant and unmet medical need to determine, predict, evaluate, and / or monitor congestion and decongestion in a quantitative and qualitative manner. There is a need to determine, predict, evaluate, and / or monitor the extent of congestion, that is, the degree of congestion.

[0038] Thus, the methods of the present invention in all embodiments of the invention allow Petition 870230018821, dated 06 / 03 / 2023, page 22 / 138 15 / 98 a) to diagnose congestion quantitatively and qualitatively in an individual and to assess or monitor the extent of congestion in an individual, b) to predict or determine or monitor the need for congestion therapy or intervention, or to predict or determine or monitor the success of a congestion therapy or intervention, or to guide a therapy or intervention in an individual, or c) to predict residual decongestion or congestion after congestion therapy or intervention in an individual or, d) to assess residual decongestion or congestion after congestion therapy or intervention in an individual; or e) to assess the decision regarding hospital discharge of an individual, where said individual is an individual having acute heart failure and / or an individual having heart failure who presents signs of worsening and / or an individual with symptoms of heart failure or acute heart failure through the use of Pro-Adrenomedullin or its fragments of at least 5 amino acids as an early surrogate marker for congestion, as described in detail in the present invention.

[0039] In particular, the methods of the present invention in all embodiments of the invention allow for the quantitative and qualitative diagnosis of congestion in an individual and assess or monitor the extent of congestion in an individual, wherein said individual has acute heart failure that is newly onset AHF or acute decompensated AHF or acute decompensated chronic AHF, or wherein said individual has chronic heart failure with signs of Petition 870230018821, dated 06 / 03 / 2023, page 23 / 138 16 / 98 worsening / symptoms of chronic heart failure wherein ProAdrenomedullin or its fragments of at least 5 amino acids is used as an early surrogate marker for congestion as described in detail in the present invention. In particular, said ProAdrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADMGly according to SEQ ID NO: 5 or CT-proADM according to SEQ ID NO: 6, preferably MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADMGly according to SEQ ID NO: 5, mature ADM-NH2 according to SEQ ID NO: 4.

[0040] In particular, the methods of the present invention in all embodiments of the invention allow predicting or determining or monitoring the need for congestion therapy or intervention or predicting or determining or monitoring the success of a congestion therapy or intervention or guiding a therapy or intervention in an individual, wherein said individual has acute heart failure that is new-onset AHF or acute decompensated HF or acute decompensated chronic HF or wherein said individual has chronic heart failure with signs of worsening / symptoms of chronic heart failure, and wherein Pro-Adrenomedullin or its fragments of at least 5 amino acids is used as an early surrogate marker for congestion as described in detail in the present invention.In particular, the aforementioned Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MR-proADM according to SEQ ID NO: 3: or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CT-proADM. Petition 870230018821, dated 06 / 03 / 2023, page 24 / 138 17 / 98 according to SEQ ID NO: 6, preferably MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5, more preferably mature ADMNH2 according to SEQ ID NO: 4.

[0041] In particular, the methods of the present invention in all embodiments of the invention allow predicting residual decongestion or congestion after congestion therapy or intervention in an individual wherein said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF, or wherein said individual has chronic heart failure with signs of worsening / symptoms of chronic heart failure and wherein Pro-Adrenomedullin or its fragments of at least 5 amino acids is used as an early surrogate marker for congestion as described in detail in the present invention.In particular, said Pro-Adrenomedullin or fragment is selected from the group comprising ProAdrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CT-proADM according to SEQ ID NO: 6, preferably MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5, more preferably ADM-NH2 according to SEQ ID NO: 4.

[0042] In particular, the methods of the present invention, in all embodiments of the invention, allow the assessment of residual decongestion or congestion after congestion therapy or intervention in an individual, wherein said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF or wherein said individual has heart failure Petition 870230018821, dated 06 / 03 / 2023, page 25 / 138 18 / 98 chronic cardiac with signs of worsening / symptoms of chronic heart failure, and in which Pro-Adrenomedullin or its fragments of at least 5 amino acids is used as an early surrogate marker for congestion as described in detail in the present invention. In particular, said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CTproADM according to SEQ ID NO: 6, preferably MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5, more preferably mature ADM-NH2 according to SEQ ID NO: 4.

[0043] In particular, the methods of the present invention in all embodiments of the invention allow the assessment of the decision regarding hospital discharge of an individual in which said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF, or in which said individual has chronic heart failure with signs of worsening / symptoms of chronic heart failure, and in which Pro-Adrenomedullin or its fragments of at least 5 amino acids is used as an early surrogate marker for congestion as described in detail in the present invention.In particular, said Pro-Adrenomedullin or fragment is selected from the group comprising ProAdrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CT-proADM according to SEQ ID NO: 6, preferably MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly. Petition 870230018821, dated 06 / 03 / 2023, page 26 / 138 19 / 98 in accordance with SEQ ID NO: 5, more preferably mature ADM-NH2 in accordance with SEQ ID NO: 4.

[0044] For the present invention, the extent of congestion can be expressed as a degree of congestion severity and has been determined as described below. However, those skilled in the art know that the extent of congestion can be expressed by other scores or substitutes, such as, for example, the score used by Ambrosy et al. (Ambrosy et al. 2013. European Heart Journal 34 (11): 835-843).

[0045] As highlighted earlier, clinical surrogates have a lower than ideal predictive value for detecting congestion. In the analysis for one of the examples shown in the present invention (PROTECT study), we combined three of the strongest clinical surrogates for congestion (i.e., JVP, peripheral edema, and orthopnea) to improve accuracy and develop a composite clinical congestion score (CCS) using the scheme presented below: Parameter 0 1 2 3 Peripheral Edema 0 Ankle Below the knee Above the knee Orthopnea 0 support point 1 support point 2 support points 3 support points JVP < 6 cm 6 to 10 cm >10 cm -

[0046] The scores for each of these three parameters were then added together to obtain a composite congestion score, which ranged from 0 to 8.

[0047] The following algorithm was then used to classify the severity of the congestion: CCS = 0, No clinical congestion CCS 1 to 3, Mild clinical congestion CCS 4 to 5, Moderate clinical congestion CCS > 6, Severe clinical congestion Petition 870230018821, dated 06 / 03 / 2023, page 27 / 138 20 / 98

[0048] In the analysis for another example shown in the present invention (BIOSTAT study), we describe the degree of congestion by the level of peripheral edema (in order of increasing severity: none, ankle, below the knee, above the knee). While PROTECT is a selected population of patients worldwide with acute decompensated heart failure, involved in a randomly chosen controlled clinical trial on the effects of rolophyllin versus placebo, BIOSTAT-CHF consists of 2 European groups of patients with signs and / or symptoms of worsening heart failure, approximately 2 / 3 were hospitalized and 1 / 3 were seen in the outpatient setting.

[0049] As explained above, congestion can be classified in many different ways. Those skilled in the art know that the extent of congestion can be expressed by other scores or surrogates. Clinical classification can be based on bedside physical examination to detect the presence of clinical symptoms / signs of congestion ('wet' vs. 'dry' if present vs. absent) and / or peripheral hypoperfusion ('cold' vs. 'warm' if present vs. absent) (for review see Ponikowski et al. 2016. Eur Heart J. ehw128). Combining these options identifies four groups: warm and wet (well perfused and congested) – most commonly present; cold and wet (hypoperfused and congested); cold and dry (hypoperfused without congestion); and warm and dry (compensated, well perfused without congestion). This classification can be useful for guiding therapy in the initial phase and carries prognostic information.

[0050] Typically, the symptoms and signs of AHF reflect fluid overload (pulmonary congestion and / or peripheral edema) or, less frequently, reduced cardiac output with peripheral hypoperfusion. Chest X-ray may be a useful test for the diagnosis of AHF. Pulmonary venous congestion, pleural effusion, interstitial edema. Petition 870230018821, dated 06 / 03 / 2023, page 28 / 138 21 / 98 or alveolar hypertrophy and cardiomegaly are the most specific findings for AHF, although in up to 20% of patients with AHF, the chest X-ray is nearly normal.

[0051] Symptoms / signs of congestion (left side) are defined as orthopnea, paroxysmal nocturnal dyspnea, wheezing (bilateral), peripheral edema (bilateral). Symptoms / signs of congestion (right side) are defined as jugular venous dilation, peripheral edema (bilateral), congested hepatocytes, hepatojugular reflux, ascites, symptoms of intestinal congestion (for review see table 12.2 in Ponikowski et al. 2016. Eur Heart J. ehw128).

[0052] Edema is an accumulation of fluid in the intercellular tissue resulting from an abnormal expansion in the volume of interstitial fluid. The fluid between the interstitial and intravascular spaces is regulated by the capillary hydrostatic pressure gradient and the oncotic pressure gradient across the capillary (Trayes et al. 2013. Am Fam Physician 88(2):102-110). Fluid accumulation occurs when local or systemic conditions disrupt this balance, leading to increased capillary hydrostatic pressure, increased plasma volume, decreased plasma oncotic pressure (hypoalbuminemia), increased capillary permeability, or lymphatic obstruction.

[0053] Clinically, edema manifests as swelling: The amount of interstitial fluid is determined by fluid homeostasis balance, and increased fluid secretion into the interstitium, or impaired fluid removal, can cause edema. An elevation in hydrostatic pressure occurs in heart failure. Causes of edema that are generalized to the whole body can cause edema in multiple organs and peripherally. For example, severe heart failure can cause pulmonary edema, pleural effusions, ascites, and peripheral edema. Petition 870230018821, dated 06 / 03 / 2023, page 29 / 138 22 / 98

[0054] Pulmonary edema is the accumulation of fluid in the air spaces and parenchyma of the lungs, leading to impaired gas exchange and potentially causing respiratory failure. It is due to either failure of the left ventricle of the heart to adequately remove blood from the pulmonary circulation (cardiogenic pulmonary edema), or damage to the lung parenchyma or lung vasculature (non-cardiogenic pulmonary edema) (Ware and Matthay 2005. N. Engl. J. Med. 353 (26): 2788-96). Treatment focuses on three aspects: first, improving respiratory function; second, treating the underlying cause; and third, preventing further lung damage. Pulmonary edema, especially acute edema, can lead to fatal respiratory distress or cardiac arrest due to hypoxia. It is a cardinal feature of congestive heart failure.

[0055] The defining symptom of pulmonary edema is difficulty breathing, but it can also include coughing up blood (classically seen as pink, frothy sputum), excessive sweating, anxiety, and pale skin. Shortness of breath can manifest as orthopnea (inability to lie down due to shortness of breath) and / or paroxysmal nocturnal dyspnea (episodes of sudden and severe shortness of breath at night). These are the common presenting symptoms of chronic pulmonary edema due to left ventricular failure.The development of pulmonary edema may be associated with symptoms and signs of fluid overload; this is a non-specific term to describe the manifestations of left ventricular failure on the rest of the body and includes peripheral edema (swelling of the legs, generally of the sulcular variety, where the skin is slow to return to normal when pressed), elevated jugular venous pressure, and hepatomegaly, where the liver is enlarged and may be tender or even pulsatile. Other signs include inspiratory end-cranes (sounds heard at the end of a deep breath) on auscultation and in the... Petition 870230018821, dated 06 / 03 / 2023, page 30 / 138 23 / 98 presence of a third heart sound.

[0056] In the present invention, it is demonstrated that ProAdrenomedullin or its fragments are associated with the severity of congestion and extent of edema, indicating decongestion and the applied dose of diuretics.

[0057] In one aspect of the present invention, residual congestion is defined as a CCS > 2 based on assessments of JVP, orthopnea, and edema near day 7.

[0058] In one aspect of the present invention, decongestion is defined by any of the following three markers of the current state of the art, alone or in combination: • Diuretic response: defined as weight loss by day 4 per 40 mg diuretic dose. • Hemoconcentration: coded as 0 (if there is a decline or no change in hemoglobin levels by day 4 compared to the reference value) or 1 (if there is an increase in hemoglobin levels by day 4 compared to the reference value). • Significant residual congestion: defined as a CCS > 2 based on assessments of JVP, orthopnea, and edema by day 7.

[0059] The subject matter of the present invention is a method for: a) to diagnose congestion or to assess or monitor the extent of congestion in an individual or, b) to predict or determine or monitor the need for congestion therapy or intervention, or to predict or determine or monitor the success of a congestion therapy or intervention, or to guide a therapy or intervention in an individual, or c) to predict residual decongestion or congestion Petition 870230018821, dated 06 / 03 / 2023, page 31 / 138 24 / 98 after congestion therapy or intervention in an individual, or d) to assess residual decongestion or congestion after congestion therapy or intervention in an individual; or e) to assess the decision regarding hospital discharge of an individual, where said individual is an individual having acute heart failure and / or an individual having heart failure who presents signs of worsening and / or an individual with symptoms of heart failure or acute heart failure through the use of ProAdrenomedullin or its fragments of at least 5 amino acids as an early surrogate marker for congestion comprising: • To determine the level of immunoreactive analyte through the use of at least one ligand that binds to a region within the amino acid sequence of Pro-Adrenomedullin or its fragments of at least 5 amino acids in a body fluid obtained from said individual; and a) correlate said level of immunoreactive analyte with the extent of congestion in said individual or diagnose congestion where a level elevated above a certain limit is indicative of congestion or the extent of congestion or, b) correlate said level of immunoreactive analyte with the need for or success of a congestion therapy or intervention in said individual, wherein a level below a certain threshold is predictive of successful congestion therapy or intervention, and wherein a level above a certain threshold is indicative of the need for congestion therapy or intervention; Petition 870230018821, dated 06 / 03 / 2023, page 32 / 138 25 / 98 c) correlate said level of immunoreactive analyte with a prediction of residual decongestion or congestion after therapy or congestion intervention, where a level elevated above a certain threshold indicates residual congestion after therapy or congestion intervention, while a level below a certain threshold indicates decongestion after therapy or, d) correlate said level of immunoreactive analyte with decongestion or residual congestion after congestion therapy or intervention, where a level elevated above a certain limit indicates residual congestion after congestion therapy or intervention, while a level below a certain limit indicates decongestion after congestion therapy or intervention, or (e) correlate said immunoreactive analyte level with the assessment of the decision regarding hospital discharge, wherein a level elevated above a certain limit means that the individual should not be discharged and wherein a level below a certain limit means that the individual may be discharged, wherein said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRproADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CTproADM according to SEQ ID NO: 6.

[0060] It can be seen from the examples that the levels in the reference value of Pro-Adrenomedullin or its fragment, in particular Petition 870230018821, dated 06 / 03 / 2023, p. 33 / 138 26 / 98 ADM-NH2, according to SEQ ID NO: 4, independently indicates significant residual congestion, for example, on day 7, diuretic response, for example, on day 4, and hemoconcentration, for example, on day 4. There are few clinical variables or biomarkers at baseline associated with the severity of congestion at the reference value, and Pro-Adrenomedullin or its fragment, in particular ADM-NH2, according to SEQ ID NO: 4, appears to be by far the strongest. Pro-Adrenomedulin or its fragment, in particular ADM-NH2 according to SEQ ID NO: 4, is strongly associated with the severity of clinical congestion at baseline and independently predicts the presence of significant residual congestion, for example, on day 7. The data strongly support the notion that Pro-Adrenomedulin or its fragment, in particular ADM-NH2 according to SEQ ID NO: 4, is a reliable, quantitative, and early surrogate for congestion.This is a fascinating finding given that there is a great unmet medical need for an accurate and reliable tool for assessing congestion in patients with AHF, particularly for ascertaining the presence of residual congestion pre-release. This, according to the present invention, Pro-Adrenomedullin or its fragment, in particular ADMNH2 according to SEQ ID NO: 4, can be used to ascertain the presence of residual congestion pre-release.

[0061] The subject matter of the present invention is a method in which said extent of congestion is expressed as a congestion score, in particular a clinical congestion score.

[0062] The subject matter of the present invention is a method by which said individual is stratified: a) in groups of congestion degrees or, b) in non-responders and / or responders and / or poor responders to congestion therapy or intervention, Petition 870230018821, dated 06 / 03 / 2023, page 34 / 138 27 / 98 c) in a decongested group or in a group with residual congestion after congestion therapy or intervention.

[0063] The subject matter of the present invention is a method in which said individual is stratified into groups of patients wherein one group comprises patients in need of therapy and the other group comprises patients who are not in need of therapy.

[0064] Previously, among others, BNP and proBNP markers were considered as congestion markers. Surprisingly, the Pro-Adrenomedullin biomarker or the respective fragments of the present invention are far superior to any other clinical or biochemical parameter used to date as a congestion marker. This was shown, for example, by Kremer et al. European Journal of Heart Failure (2017) 19 (Suppl S1) 5601, Kremer et al. studied the potential role of bio-ADM, a biomarker representing biologically active adrenomedullin, as a potential congestion marker in ADHF, and assessed the added prognostic value beyond clinically assessed congestion. Bio-ADM was assessed at baseline, days 2 and 7 in 1562 patients admitted for ADHF. Clinical congestion was assessed using a composite clinical congestion score (CCS) encompassing edema, orthopnea, and jugular venous distension.This was compared with other clinical and biochemical parameters, including BNP. Among a large number of clinical and biochemical parameters, bio-ADM was the strongest predictor of clinically assessed congestion. Bio-ADM levels at the reference value, but not BNP levels, were strongly and independently associated with the presence of significant residual congestion on day 7. In patients with residual congestion on day 7, bio-ADM levels remained high, while BNP levels decreased. Bio-ADM, but not BNP, Petition 870230018821, dated 06 / 03 / 2023, page 35 / 138 28 / 98 was a strong independent predictor of 60-day heart failure rehospitalization, in addition to clinically assessed congestion. Thus, the authors concluded that bio-ADM is elevated in patients with ADHF and is a promising marker of (residual) congestion in patients admitted for ADHF. Bio-ADM was a strong and independent predictor of early rehospitalization after discharge.

[0065] In a specific embodiment of the invention, said proADM and / or its fragments having at least 5 amino acids are selected from the group comprising:

[0066] SEQ ID NO: 1 (proADM): 164 amino acids (22 to 185 from preproADM) ARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

[0067] SEQ ID NO: 2 (ProAdrenomedullin N-20 terminal peptide, PAMP): amino acids 22 to 41 of preproADM

[0068] ARLDVASEF RKWNKWALS R

[0069] SEQ ID NO: 3 (Medium Regional Pro-Adrenomedullin, MRproADM): amino acids 45 to 92 of preproADM

[0070] ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV

[0071] SEQ ID NO: 4 (Mature adrenoedulin (mature ADM); ADM converted to amide; bio-ADM; hADM): amino acids 95 to 146 -CONH2

[0072] YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGY - CONH2

[0073] SEQ ID NO: 5 (Adrenomedullin 1-52-Gly (ADM 1-52-Gly) amino acids 95 to 147 of preproADM

[0074] YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK Petition 870230018821, dated 06 / 03 / 2023, page 36 / 138 29 / 98 DKDNVAPRSK ISPQGYG

[0075] SEQ ID NO: 6 (C-terminal Pro-Adrenomedullin, CTproADM): amino acids 148 to 185 of preproADM

[0076] RRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

[0077] In a specific embodiment of the invention, said proADM and / or its fragments having at least 5 amino acids are selected from the group comprising ADM-NH2 (SEQ ID NO: 4), ADM 1-52-Gly (SEQ ID NO: 5), MR-proADM (SEQ ID NO: 3) and CTproADM (SEQ ID NO: 6).

[0078] In a specific embodiment of the invention, the level of mature ADM-NH2 (SEQ ID NO: 4) and / or ADM 1-52-Gly (SEQ ID NO: 5) immunoreactivity or the level of MR-proADM (SEQ ID NO: 3) immunoreactivity or the level of CT-proADM (SEQ ID NO: 6) immunoreactivity is determined and correlated with the need of said patient for therapy or intervention, wherein said patient is identified as having such a need if the level of mature ADM-NH2 (SEQ ID NO: 4) and / or ADM 1-52-Gly (SEQ ID NO: 5) immunoreactivity or the level of MR-proADM (SEQ ID NO: 3) immunoreactivity or the level of CT-proADM (SEQ ID NO: 6) immunoreactivity in the body fluid of said individual is above a threshold.

[0079] In a specific embodiment of the invention, the level of proADM and / or its fragments is determined by the use of at least one ligand selected from the group: a ligand that binds to a region comprised within the following sequence of ADM-NH2 (SEQ ID NO: 4) and / or ADM 1-52-Gly (SEQ ID NO: 5) and a second ligand that binds to a region comprised within the mature ADM-NH2 sequence (SEQ ID NO: 4) and / or ADM 1-52-Gly (SEQ ID NO: 5).

[0080] In a specific embodiment of the invention, the level of proADM and / or its fragments is determined by the use of at least one ligand selected from the group: a ligand that binds to a region comprised within the MR-proADM sequence (SEQ ID NO: 3) and Petition 870230018821, dated 06 / 03 / 2023, page 37 / 138 30 / 98 a second ligand that binds to a region within the MR-proADM sequence (SEQ ID NO: 3).

[0081] In a specific embodiment of the invention, the level of proADM and / or its fragments is determined by the use of at least one ligand selected from the group: a ligand that binds to a region comprised within the CT-proADM sequence (SEQ ID NO: 6) and a second ligand that binds to a region comprised within the CT-pro-ADM sequence (SEQ ID NO: 6).

[0082] The matter in a particular embodiment of the present invention is a method, according to the present invention, whereby said fragment can be selected from MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4.

[0083] The subject matter of the present invention is a method according to the present invention, in which the level of Pro-Adrenomedullin or its fragments of at least 5 amino acids is determined by the use of a ligand for Pro-Adrenomedullin or its fragments of at least 5 amino acids.

[0084] The subject matter of the present invention is a method, according to the present invention, in which the ligand is selected from the group comprising an antibody, antibody fragment or a non-Ig support linkage to Pro-Adrenomedullin or its fragments of at least 5 amino acids.

[0085] A body fluid according to the present invention is in a particular embodiment of a blood sample. A blood sample may be selected from the group comprising whole blood, serum and plasma. In a specific embodiment of the invention, said sample is selected from the group comprising human citrate plasma, heparin plasma and EDTA plasma.

[0086] The subject matter of the present invention is a method, according to the present invention, in which said determination of Petition 870230018821, dated 06 / 03 / 2023, page 38 / 138 31 / 98 Adrenomedulina or its fragments of at least 5 amino acids is run more than once in a patient.

[0087] The subject matter of the present invention is a method in which the sample is taken at hospital admission or before hospital discharge.

[0088] The subject matter of the present invention is a method, according to the present invention, in which said monitoring is performed in order to evaluate the response of said individual to the preventive and / or therapeutic measures taken.

[0089] The subject matter of the present invention is a method, according to the present invention, in which said method is used to stratify said individuals into groups according to degree of congestion.

[0090] The subject matter of the present invention is a method according to the present invention in which said level of Pro-Adrenomedullin or its fragments is correlated with a risk of death or an adverse event in an individual with acute heart failure or heart failure, wherein a level elevated above a certain threshold is prognostic of a higher risk of death or adverse events.

[0091] In a specific embodiment of the invention, an assay is used to determine the level of proADM and / or its fragments having at least 5 amino acids, wherein the sensitivity of said assay is capable of quantifying mature ADM-NH2 from healthy individuals and is < 70 pg / mL, preferably < 40 pg / mL and more preferably < 10 pg / mL.

[0092] In a specific embodiment of the invention, an assay is used to determine the level of proADM and / or its fragments having at least 5 amino acids, wherein the sensitivity of said assay is capable of quantifying the MR-proADM of healthy individuals and is < 0.5 nmol / L, preferably < 0.4 nmol / L and more preferably < 0.2 nmol / L.

[0093] In a specific embodiment of the invention, an assay is used to determine the level of proADM and / or its fragments having Petition 870230018821, dated 06 / 03 / 2023, p. 39 / 138 32 / 98 at least 5 amino acids, wherein the sensitivity of said assay is capable of quantifying CT-proADM of healthy individuals and is < 100 pmol / L, preferably < 75 pmol / L and more preferably < 50 pmol / L.

[0094] In a specific embodiment of the invention, said ligand exhibits a binding affinity to proADM and / or its fragments of at least 107 M-1, preferably 108 M-1, the preferred affinity is greater than 109 M-1, most preferably greater than 1010 M-1. A person skilled in the art knows that it may be considered to compensate for the lower affinity by applying a higher dose of compounds and this measure would not lead outside the scope of the invention.

[0095] To determine the affinity of antibodies for the Adrenomedullin, the binding kinetics of Adrenomedullin to the immobilized antibody was determined by means of marker-free surface plasmon resonance, using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of antibodies was performed using an Fc anti-mouse antibody covalently coupled at high density to a CM5 sensor surface according to the manufacturer's instructions (mouse antibody capture kit; GE Healthcare), (Lorenz et al. 2011. Antimicrob Agents Chemother. 55 (1): 165-173).

[0096] In a specific embodiment of the invention, said ligand is selected from the group comprising an antibody or an antibody fragment or a non-Ig structure that binds to proADM and / or its fragments.

[0097] In a specific embodiment of the invention, an assay is used to determine the level of proADM and / or its fragments having at least 5 amino acids, wherein such assay is an interleaved assay, preferably a fully automated assay. Petition 870230018821, dated 06 / 03 / 2023, page 40 / 138 33 / 98

[0098] In one embodiment of the invention, it may be a so-called POC (point-of-care) test, which is a testing technology that allows the test to be performed within less than an hour near the patient without the need for a fully automated assay system. An example of this technology is the immunochromatographic test technique.

[0099] In one embodiment of the invention, such an assay is an intercalated immunoassay that utilizes any type of detection technology including, but not limited to, enzyme labeling, chemiluminescence labeling, electrochemiluminescence labeling, preferably a fully automated assay. In one embodiment of the invention, such an assay is an enzyme-labeled intercalated assay. Examples of automated or fully automated assays include assays that can be used for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, BiomerieuxVidas®, Alere Triage®.

[00100] A variety of immunoassays are known and can be used for the assays and methods of the present invention, these include: radioimmunoassays (RIA), enzyme-multiplied homogeneous immunoassays (EMPT), enzyme-linked immunosorbent assays (ELISA), apoenzyme reactivation immunoassay (ARIS), probe immunoassays and immunochromatographic assays.

[00101] In a specific embodiment of the invention, at least one of said two ligands is labeled in order to be detected.

[00102] Preferred detection methods include immunoassays in various formats, such as, for example, radioimmunoassay (RIA), chemiluminescence and fluorescence immunoassays, enzyme-linked immunosorbent assays (ELISA), Luminex-based globule arrays, protein microarray assays, and rapid test formats such as, for example, strip tests. Petition 870230018821, dated 06 / 03 / 2023, p. 41 / 138 34 / 98 immunochromatographic.

[00103] In a preferred embodiment, said marker is selected from the group comprising a chemiluminescent marker, enzyme marker, fluorescent marker, radioiodine marker.

[00104] Assays can be homogeneous or heterogeneous, competitive and non-competitive. In one embodiment, the assay is in the form of an intercalated assay, which is a non-competitive immunoassay, where the molecule to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase, for example, a globule, the surface of a reservoir or other container, a chip or a strip, and the second antibody is an antibody that is labeled, for example, with a dye, a radioisotope, or a reactive or catalytically active component. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedures involved with intercalated assays are well established and known to those versed in the field (The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005); Hultschig et al. 2006. Curr Opin Chem Biol.10 (1):4-10).

[00105] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present as dispersions in a liquid reaction mixture, wherein a first labeling component is bound to the first capture molecule, wherein said first labeling component is part of a labeling system based on fluorescence quenching or chemiluminescence or amplification, and a second labeling component of said labeling system is attached to the second capture molecule, such that after binding of both capture molecules to the analyte, a measurable signal is generated which accounts for the detection of sandwich complexes formed in the solution comprising the sample. Petition 870230018821, dated 06 / 03 / 2023, page 42 / 138 35 / 98

[00106] In another embodiment, said labeling system comprises rare earth cryptates or rare earth chelates in combination with a fluorescent dye or chemiluminescent dye, in particular a cyanine-type dye.

[00107] In the context of the present invention, fluorescence-based assays comprise the use of dyes, which may, for example, be selected from the group comprising FAM (5- or 6-carboxyfluorescein), VIC, NAD, Fluorescein, Fluoresceinisothiocyanate (FITC), IRD-700 / 800, Cyanine dyes such as CY3, CY5, CY35, CY5.5, CY7, Xanthein, 6-Carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET, 6-Carboxy-4',5'-dichloro-2',7'-dimethodifluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrodamine (TAMRA), 6-Carboxy-Xrhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine6G (RG6), Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes, such as BODIPY TMR, Oregon Green, Coumarins such as Umbelliferone, Benzimides, such as Hoechst 33258; Phenanthridines, such as Texas Red, Yakima Yellow, Alexa Fluor, PET, Ethidium Bromide, Acridinium dyes, Carbazole dyes, Phenoxazine dyes, Porphyrin dyes, Polymethine dyes, and others.

[00108] In the context of the present invention, chemiluminescence-based assays comprise the use of dyes, based on the physical principles described for chemiluminescent materials in (Kirk-Othmer, Encyclopedia of chemical technology, 4th ed. 1993. John Wiley & Sons, Vol. 15: 518-562, incorporated herein by reference, including citations on pages 551-562). Preferred chemiluminescent dyes are acridinium esters.

[00109] As mentioned in this article, a diagnostic assay or assay can be of any type applied in the field of diagnostics. Such an assay may be based on the binding of an analyte. Petition 870230018821, dated 06 / 03 / 2023, p. 43 / 138 36 / 98 to be detected by one or more capture probes with a certain affinity. Concerning the interaction between capture molecules and target molecules or molecules of interest, the affinity constant is preferably greater than 108M-1.

[00110] In the context of the present invention, binding molecules are molecules that can be used to bind target molecules or molecules of interest, i.e., analytes (i.e., in the context of the present invention, ADM-NH2 and / or proADM and their fragments), from a sample. The binding molecules must thus be suitably formed, both spatially and in terms of surface characteristics, such as surface charge, hydrophobic capacity, hydrophilic capacity, presence or absence of Lewis donors and / or acceptors, to specifically bind the target molecules or molecules of interest. For example, binding can be mediated by ionic, van der Waals, pi-pi, sigma-pi, hydrophobic, or hydrogen bonding interactions, or a combination of two or more of the aforementioned interactions between the binding molecules and the target molecules or molecules of interest.In the context of the present invention, the linking molecules may, for example, be selected from the group comprising a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, an antibody, a peptide, or a glycoprotein. Preferably, the linking molecules are antibodies, including their fragments, with sufficient affinity for a target or molecule of interest, and including recombinant antibodies or fragments of recombinant antibodies, as well as chemically and / or biochemically modified derivatives of said antibodies or fragments derived from the variant chain with a length of at least 12 amino acids.

[00111] The chemiluminescent marker can be a marker of Petition 870230018821, dated 06 / 03 / 2023, page 44 / 138 37 / 98 acridinium ester, steroid markers involving isoluminol markers and others.

[00112] Enzyme markers can be lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase, glucose-6-phosphonate dehydrogenase, and so on.

[00113] In one embodiment of the invention, at least one of said two binders is attached to a solid phase such as magnetic particles and polystyrene surfaces.

[00114] In a specific embodiment of the invention, at least one of said two binders is attached to a solid phase.

[00115] In a specific embodiment of the invention, the limit is within a limit range for plasma ADM-NH2 that is between 50 and 100 pg / mL, preferably between 60 and 90 pg / mL, most preferably a limit of 70 pg / mL is applied.

[00116] In a specific embodiment of the invention, the limit is within a limiting range for plasma MR-proADM that is between 0.5 and 1.5 nmol / L, preferably between 0.7 and 1 nmol / L, most preferably a limit of 0.8 nmol / L is applied.

[00117] In a specific embodiment of the invention, the limit is within a limiting range for plasma CT-proADM that is between 85 and 350 pmol / L, preferably between 100 and 250 pmol / L, most preferably a limit of 150 pmol / L is applied.

[00118] The ADM-NH2 levels of the present invention, or the levels of proADM or its fragments, respectively, were determined using the ADM-NH2 assay described, as outlined in the examples (or assays of proADM or its fragments, respectively). The threshold values ​​mentioned above may differ in other assays if these were calibrated differently from the assay systems used in the present invention. Therefore, the cutoff values Petition 870230018821, dated 06 / 03 / 2023, p. 45 / 138 38 / 98 mentioned above should apply to such differently calibrated assays, consequently taking into account the differences in calibration. One possibility for quantifying the difference in calibration is a comparative analysis (correlation) of the assay method in question with the respective biomarker assay used in the present invention, by measuring the respective biomarker (e.g., bio-ADM) in samples using both methods. Another possibility is to determine, with the assay in question, given that this test has sufficient analytical sensitivity, the average biomarker level of a representative normal population, compare the results with the average biomarker levels as described in the literature, and recalculate the calibration based on the difference obtained by this comparison.With the calibration used in the present invention, samples from normal (healthy) individuals were measured: median plasma bioADM (mature NH2-ADM) was 24.7 pg / mL, the lowest value was 11 pg / mL, and the 99th percentile was 43 pg / mL. Alternatively, commercially available control samples can be used to adjust different calibrations (e.g., ICI Diagnostics, Berlin, Germany).

[00119] The mean plasma MR-proADM concentration in normal (healthy) individuals was 0.41 (interquartile range 0.23 to 0.64) nmol / L (Smith et al. 2009. Clin Chem 55:1593-1595) using the automated intercalated fluorescence assay for MR-proADM detection as described above in Caruhel et al. (Caruhel et al. 2009. Clin Biochem 42:725-8).

[00120] The mean plasma concentration of CT-proADM in normal healthy subjects (n = 200) was 77.6 pmol / L (min 46.6 pmol / L, max 136.2 pmol / L) and the 95th percentile was 113.8 pmol / L (SE 2 111 552 B1).

[00121] In a specific embodiment of the invention, a limit for Petition 870230018821, dated 06 / 03 / 2023, p. 46 / 138 39 / 98 The ADM-NH2 in plasma is the 5-times average concentration, preferably the 4-times median concentration, most preferably the three-times average concentration, most preferably the two-times average concentration of a normal healthy population.

[00122] In a specific embodiment of the invention, a limit for MR-proADM in plasma is the 5-times average concentration, preferably the 4-times average concentration, most preferably the three-times average concentration, most preferably the two-times average concentration of a normal healthy population.

[00123] In a specific embodiment of the invention, a limit for CT-proADM in plasma is the 5-times average concentration, preferably the 4-times average concentration, most preferably the three-times average concentration, most preferably the two-times average concentration of a normal healthy population.

[00124] An antibody according to the present invention is a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes that bind specifically to an antigen. Recognized immunoglobulin genes include the constant region genes kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM), as well as the numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally around 25 kDa or 214 amino acids in length. Full-length immunoglobulin heavy chains are generally around 50 kDa or 446 amino acids in length. The light chains are encoded by a variable region gene at the NH2 terminus (approximately 110 amino acids in length) and by a constant region gene kappa or lambda at the COOH terminus.The heavy chains are similarly encoded by a variable region gene (approximately 116 amino acids long) and one of the other constant region genes.

[00125] The basic structural unit of an antibody is generally Petition 870230018821, dated 06 / 03 / 2023, page 47 / 138 40 / 98 is a tetramer consisting of two identical pairs of immunoglobulin chains, each pair having a light chain and a heavy chain. In each pair, the variable regions of the light and heavy chains bind to an antigen, and the constant regions act as mediators of effector functions. Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al. 1987. Eur. J. Immunol. 17:105; Huston et al. 1988, Proc. Natl. Acad. Sci. USA, 85:5879-5883; Bird et al. 1988, Science 242:423-426; Hood et al., Immunology, Benjamin, NY, 2nd ed., 1984; Hunkapiller and Hood 1986. Nature 323:15-16). A variable region of an immunoglobulin light or heavy chain includes a region of interrupted structure by three hypervariable regions, also called complementarity-determining regions (CDRs) (see, Sequences of Proteins of Immunological Interest, E.(Kabatet al., US Department of Health and Human Services, 1983). As noted above, CDRs are primarily responsible for binding to an epitope of an antigen. An immune complex is an antibody, such as a monoclonal antibody, chimeric antibody, humanized antibody, or human antibody, or a functional antibody fragment, specifically bound to the antigen.

[00126] Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically through genetic engineering, from genes in the variable and constant regions of immunoglobulins belonging to different species. For example, variable gene segments from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3. In one example, a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from an antibody. Petition 870230018821, dated 06 / 03 / 2023, p. 48 / 138 41 / 98 of mouse and the constant or effector domain from a human antibody, although other mammalian species may be used, or the variable region may be produced by molecular techniques. Methods for producing chimeric antibodies are well known in the art (e.g., see US Patent 5,807,715). A humanized immunoglobulin is an immunoglobulin that includes a human structure region and one or more CDRs from a non-human immunoglobulin (such as a mouse, rat, or synthetic immunoglobulin). The non-human immunoglobulin that provides the CDRs is called a donor, and the human immunoglobulin that provides the structure is called an acceptor. In one embodiment, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin.The constant regions do not need to be present, but if they are, they must be substantially identical to the constant regions of human immunoglobulin, that is, at least about 85 to 90%, such as around 95% or more identical. Therefore, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized light chain immunoglobulin and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor structure of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions taken from the donor structure.Humanized monoclonal antibodies or others may have additional conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are those such as gly, ala; val, ile, leu;. Petition 870230018821, dated 06 / 03 / 2023, p. 49 / 138 42 / 98 asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed through genetic engineering (e.g., see US Patent 5,585,089). A human antibody is an antibody in which the light and heavy chain genes are of human origin. Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell that secretes the antibody of interest. Immortization can be accomplished, for example, through EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a triome cell. Human antibodies can also be produced by phage presentation methods (see, for example, Dower et al., PCT Publication No. WO91 / 17271; McCafferty et al., PCT Publication No. WO92 / 001047; and Winter, PCT Publication No.WO92 / 20791), or selected from a library of combinatorial human monoclonal antibodies (see the Morphosys website). Human antibodies can also be prepared using transgenic animals carrying a human immunoglobulin gene (e.g., see Lonberg et al., PCT Publication No. WO93 / 12227; and Kucherlapati, PCT Publication No. WO91 / 10741).

[00127] Thus, the antibody can have the shapes known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies. In a preferred embodiment, the antibodies according to the present invention are recombinantly produced antibodies, for example, IgG, a typical full-length immunoglobulin, or antibody fragments containing at least the F-variable heavy and / or light chain domain, for example, chemically coupled antibodies (fragment antigen binding) including, but not limited to, Fab fragments that include Fab minibodies, Fab antibody Petition 870230018821, dated 06 / 03 / 2023, page 50 / 138 43 / 98 single-chain, monovalent Fab antibody with epitope markers, for example, Fab-V5Sx2; bivalent Fab (mini-antibody) dimerized with the CH3 domain; bivalent or multivalent Fab, for example, formed by multimerization with the aid of a heterologous domain, for example, by dimerization of dHLX domains, for example Fab-dHLX-FSx2; F(ab')2 fragments, scFv fragments, multivalent and / or multispecific scFv fragments, bivalent and / or bispecific diabodies, BITE® (bispecific T cell occupier), tri-functional antibodies, polyvalent antibodies, for example, of a different G class; single-domain antibodies, for example, nanobodies derived from camelid or fish immunoglobulins and numerous others.

[00128] In addition to antibodies, other biopolymer structures are well known in the art for mixing to form a complex with a target molecule and have been used for the generation of highly targeted specific biopolymers. Examples are aptamers, spiegelmers, antigens, and conotoxins.

[00129] In a preferred embodiment, the antibody format is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2 fragment, and scFv-Fe fusion protein. In another preferred embodiment, the antibody format is selected from the group comprising scFab fragment, Fab fragment, scFv fragment, and their bioavailability-optimized conjugates, such as Pegylated fragments. One of the most preferred formats is the scFab format.

[00130] Non-Ig structures can be structural proteins and can be used as antibody mimetics since they are capable of binding to ligands or antigens. Non-Ig structures can be selected from the group comprising tetranectin-based non-Ig structures (e.g., described in US 2010 / 0028995), structures Petition 870230018821, dated 06 / 03 / 2023, page 51 / 138 44 / 98 fibronectin (e.g., described in EP 1 266 025), lipocalin-based structures (e.g., described in WO 2011 / 154420): ubiquitin structures (e.g., described in WO 2011 / 073214), transfer structures (e.g., described in US 2004 / 0023334), protein A structure (e.g., described in EP 2 231 860), ankyrin repeat-based structures (e.g., described in WO 2010 / 060748), microprotein structures (preferably microproteins forming a cystine loop) (e.g., described in EP 2 314 308), Fyn SH3 domain-based structures, frames (e.g., described in WO 2011 / 023685), EGFR-A domain-based structures (e.g., described in WO 2005 / 040229) and structures based on the Kunitz domain (e.g., described in EP 1941867).

[00131] In one embodiment of the invention, the antibodies according to the present invention can be produced as follows.

[00132] A Balb / c mouse was immunized with ADM-100 μg of peptide-BSA conjugate on days 0 and 14 (emulsified in 100 pL of complete Freund's adjuvant) and 50 pg on days 21 and 28 (in 100 pL of incomplete Freund's adjuvant). Three days before the fusion experiment was performed, the animal received 50 pg of the conjugate dissolved in 100 pL of saline solution, administered as an intraperitoneal injection and an intravenous injection.

[00133] Splenocytes from immunized mice and SP2 / 0 myeloma cell line cells were fused with 1 mL of 50% polyethylene glycol for 30 s at 37°C. After washing, the cells were seeded in 96-pool cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement]. After two weeks, the HAT medium was replaced with HT medium for three passages followed by return. Petition 870230018821, dated 06 / 03 / 2023, page 52 / 138 45 / 98 for normal cell culture medium.

[00134] Cell culture supernatants were subjected to primary screening for antigen-specific IgG antibodies three weeks after fusion. Microcultures that tested positive were transferred into 24-well plates for propagation. After repeat testing, selected cultures were cloned and cloned again using the limited dilution technique and isotypes were determined (see also Lane 1985. J. Immunol. Meth. 81: 223-228; Ziegler, B. et al. 1996 Horm. Metab. Res. 28: 11-15).

[00135] Antibodies can be produced via phage presentation according to the following procedure.

[00136] Human free antibody gene libraries HAL7 / 8 were used for the isolation of recombinant single-chain F-variable domains (scFv) against the adrenomedullin peptide. Antibody gene libraries were screened using an extraction strategy comprising the use of peptides containing a biotin tag linked via two different spacers in the adrenomedullin peptide sequence. A mixture of extraction rounds using the non-specifically bound antigen and the streptavidin-bound antigen was used to minimize the formation of non-specific ligands. Phages eluted from the third extraction round were used to generate E. coli strains expressing monoclonal scFv. The culture supernatant from these clonal strains was used directly for an antigen ELISA assay (see Hust et al. 2011. Journal of Biotechnology 152: 159-170; Schütte et al. 2009. PLoS One 4, e6625).

[00137] The humanization of murine antibodies can be conducted according to the following procedure.

[00138] For the humanization of a murine-derived antibody, the antibody sequence is analyzed with respect to the structural interaction of the structural regions (FR) with the determinant regions. Petition 870230018821, dated 06 / 03 / 2023, p. 53 / 138 Complementary 46 / 98 (CDR) and the antigen. Based on structural modeling, an appropriate human-derived FR is selected, and murine CDR sequences are transplanted into the human FR. Variations in the amino acid sequence of CDRs or FRs can be introduced to recover structural interactions that were abolished by the species switch for FR sequences. This recovery of structural interactions can be achieved through a random approach using phage presentation libraries or through a targeted approach guided by molecular modeling (Almagro et al. 2008. Front Biosci. 2008; 13:1619-33).

[00139] The subject matter of the invention is also a method according to the present invention, wherein said level of Pro-Adrenomedullin or its fragments is used for guidance of therapy or intervention, wherein the therapy or intervention is indicated if said level of Pro-Adrenomedullin or fragments is above a certain limit and wherein the therapy or intervention is not indicated if said level of Pro-Adrenomedullin or fragments is below a certain limit. The limit and limit ranges are provided above.

[00140] The following embodiments are the subject of the present invention:

[00141] 1. A method for a) to diagnose congestion or to assess or monitor the extent of congestion in an individual or, b) to predict or determine or monitor the need for congestion therapy or intervention, or to predict or determine or monitor the success of a congestion therapy or intervention, or to guide a congestion therapy or intervention in an individual, or c) to predict decongestion or residual congestion after congestion therapy or intervention in an individual. Petition 870230018821, dated 06 / 03 / 2023, page 54 / 138 47 / 98 individual or, d) to assess residual decongestion or congestion after congestion therapy or intervention in an individual or, e) to assess the decision regarding hospital discharge of an individual, where said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF, or where said individual has chronic heart failure with worsening signs / symptoms of chronic heart failure and where the use of Pro-Adrenomedullin or its fragments of at least 5 amino acids is used as an early surrogate marker for congestion, comprising: • Determine the level of Pro-Adrenomedullin or its fragments of at least 5 amino acids in a body fluid obtained from said individual; and a) Correlate said level of Pro-Adrenomedullin or its fragments with the extent of congestion in said individual or diagnose congestion where a level elevated above a certain limit is indicative of congestion, or the extent of congestion or, b) Correlate said level of Pro-Adrenomedullin or its fragments with the success of a congestion therapy or intervention in said individual, wherein a level below a certain threshold is predictive of successful congestion therapy or intervention, and wherein a level above a certain threshold is indicative of the need for congestion therapy or intervention, or Petition 870230018821, dated 06 / 03 / 2023, page 55 / 138 48 / 98 c) Correlate said level of Pro-Adrenomedullin or its fragments with the prognosis of decongestion or residual congestion after therapy or congestion intervention, where a level elevated above a certain limit indicates residual congestion after therapy or congestion intervention, while a level below a certain limit indicates decongestion after therapy or congestion intervention, or d) Correlate said level of Pro-Adrenomedullin or its fragments with decongestion or residual congestion after therapy or congestion intervention, where a level elevated above a certain limit indicates residual congestion after therapy or congestion intervention, while a level below a certain limit indicates decongestion after therapy or congestion intervention, or (e) Correlate said level of Pro-Adrenomedullin or its fragments with the assessment of the decision on hospital discharge, wherein a level elevated above a certain limit means that the individual would not be discharged and wherein a level below a certain limit means that the individual may be discharged, wherein said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRproADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CT-proADM according to SEQ ID NO: 6.

[00142] 2. A method for a) to diagnose congestion or to assess or monitor the extent of congestion in an individual or, Petition 870230018821, dated 06 / 03 / 2023, page 56 / 138 49 / 98 b) to predict or determine or monitor the need for congestion therapy or intervention, or to predict or determine or monitor the success of a congestion therapy or intervention, or to guide a congestion therapy or intervention in an individual, or c) to predict residual decongestion or congestion after congestion therapy or intervention in an individual or, d) to assess residual decongestion or congestion after congestion therapy or intervention in an individual or, e) to assess the decision regarding hospital discharge of an individual, wherein said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF, or wherein said individual has chronic heart failure with worsening signs / symptoms of chronic heart failure, and wherein ProAdrenomedullin or its fragments of at least 5 amino acids are used as an early surrogate marker for congestion, comprising: • To determine the level of immunoreactive analyte through the use of at least one ligand that binds to a region within the amino acid sequence of Pro-Adrenomedullin or its fragments of at least 5 amino acids in a body fluid obtained from said individual; and a) Correlate said level of immunoreactive analyte with the extent of congestion in said individual or diagnose congestion where a level elevated above a certain limit is indicative of congestion or the extent of congestion or, Petition 870230018821, dated 06 / 03 / 2023, page 57 / 138 50 / 98 b) Correlate said level of immunoreactive analyte with the success of a congestion therapy or intervention in said individual, where a level below a certain threshold is predictive of therapy or intervention success, and where a level above a certain threshold is indicative of the need for congestion therapy or intervention. c) Correlate said level of immunoreactive analyte with a prognosis of decongestion or residual congestion after therapy or congestion intervention, where a level elevated above a certain limit indicates residual congestion after therapy or congestion intervention, while a level below a certain limit indicates decongestion after therapy or d) Correlate said level of immunoreactive analyte with decongestion or residual congestion after therapy or congestion intervention, where a level elevated above a certain limit indicates residual congestion after therapy or congestion intervention, while a level below a certain limit indicates decongestion after therapy or congestion intervention. e) Correlate said level of immunoreactive analyte with the assessment of the decision regarding hospital discharge, where a level elevated above a certain limit means that the individual would not be discharged, and where a level below a certain limit means that the individual would not be discharged, where said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRPetition 870230018821, dated 06 / 03 / 2023, page 58 / 138 51 / 98 proADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CTproADM according to SEQ ID NO: 6.

[00143] 3. A method in accordance with items 1 or 2, in which the extent of congestion is expressed as a congestion score, in particular a clinical congestion score.

[00144] 4. A method in accordance with any of items 1 to 3, in which said individual is stratified a) in groups of congestion degrees or, b) in non-responders and / or responders and / or poor responders to congestion therapy or intervention, or c) in a decongested group or in a group with residual congestion after congestion therapy or intervention.

[00145] 5. A method in accordance with any of items 1 to wherein said fragment may be selected from MR-proADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4.

[00146] 6. A method in accordance with any of items 1 to 5, wherein the level of Pro-Adrenomedullin or its fragments of at least 5 amino acids is determined by using a ligand for Pro-Adrenomedullin or its fragments of at least 5 amino acids.

[00147] 7. A method according to item 6, wherein the ligand is selected from the group comprising an antibody, an antibody fragment or a non-Ig structure that binds to Pro-Adrenomedullin or its fragments of at least 5 amino acids.

[00148] 8. A method in accordance with any of the preceding items, wherein said limit is within a limit range that is a limit range for plasma ADM-NH2 according to SEQ ID NO: 4 between 50 and 100 pg / mL and for plasma MR-proADM between 0.5 and 1.5 nmol / L, and Petition 870230018821, dated 06 / 03 / 2023, page 59 / 138 52 / 98 for plasma CT-proADM between 85 and 350 pmol / L.

[00149] 9. A method in accordance with any of items 1 to 8, where the aforementioned determination of Pro-Adrenomedullin or its fragments of at least 5 amino acids is performed more than once in a patient.

[00150] 10. A method in accordance with any of items 1 to 9, in which the sample is taken upon admission to a hospital or before hospital discharge.

[00151] 11. A method in accordance with any of items 1 to 10, in which said monitoring is carried out in order to evaluate the response of said individual to the preventive and / or therapeutic measures taken.

[00152] 12. A method according to any one of the items from 1 to a in order to stratify said individuals into groups according to degree of congestion.

[00153] 13. A method according to any one of the items from 1 to 12, wherein said level of Pro-Adrenomedullin or its fragments or said level of immunoreactive analyte is correlated with a risk of death or an adverse event in an individual who has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF or wherein said individual is an individual who has chronic heart failure with worsening signs / symptoms of chronic heart failure, wherein an elevated level above a certain threshold is predictive of an increased risk of death or adverse events.

[00154] 14. A method in accordance with any of items 1 to 13, wherein said level of Pro-Adrenomedullin or its fragments or said level of immunoreactive analyte is used to guide therapy or intervention, wherein therapy or intervention is indicated if said level of Pro-Adrenomedullin or fragments is above a certain limit and wherein therapy or intervention is not indicated if said level of Pro-Adrenomedullin or fragments is below a certain limit. Petition 870230018821, dated 06 / 03 / 2023, page 60 / 138 53 / 98

[00155] 15. A method in accordance with any of items 1 to 1 in which said level of Pro-Adrenomedullin or its fragments or said level of immunoreactive analyte is used to determine the need for therapy or intervention.

[00156] 16. A method in accordance with any of the items in items 1 to 1 in which said intervention or therapy is selected from the group comprising the administration of diuretics, the administration of inotropes, the administration of vasodilators, ultrafiltration.

[00157] 17. Use of a ligand in the manufacture of a reagent or a medicine or a kit for a) to diagnose congestion or to assess or monitor the extent of congestion in an individual or, b) to predict or determine or monitor the need for therapy or intervention or to predict or determine or monitor the success of a congestion therapy or intervention or to guide a therapy or intervention in an individual or, c) to predict residual decongestion or congestion after congestion therapy or intervention in an individual or, d) to assess residual decongestion or congestion after congestion therapy or intervention in an individual or, e) to assess the decision regarding the hospital discharge of an individual, where said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF, or where said individual has chronic heart failure with worsening signs / symptoms of chronic heart failure, and where the ligand is Petition 870230018821, dated 06 / 03 / 2023, page 61 / 138 54 / 98 binds to Pro-Adrenomedullin or its fragments of at least 5 amino acids and can determine the level of Pro-Adrenomedullin or its fragments of at least 5 amino acids in a sample of body fluid obtained from said individual, and Pro-Adrenomedullin or its fragments of at least 5 amino acids are used as an early surrogate marker for congestion; and wherein a) said level of Pro-Adrenomedullin or its fragments is correlated with the extent of congestion in said individual or diagnosis of congestion, where a level elevated above a certain threshold is indicative of congestion or, b) said level of Pro-Adrenomedullin or its fragments is correlated with the success of a therapy or intervention in said individual, wherein a level below a certain threshold is predictive of therapy or intervention success, and wherein a level above a certain threshold is indicative of the need for therapy or intervention; c) said level of Pro-Adrenomedullin or its fragments is correlated with a prognosis of decongestion or residual congestion after therapy or congestion intervention, where a level elevated above a certain threshold indicates residual congestion after therapy or congestion intervention, while a level below a certain threshold indicates decongestion after therapy or congestion intervention, or d) said level of Pro-Adrenomedullin or its fragments is correlated with decongestion or residual congestion after congestion therapy or intervention, in Petition 870230018821, dated 06 / 03 / 2023, page 62 / 138 55 / 98 that a level elevated above a certain limit indicates residual congestion after therapy or congestion intervention, while a level below a certain limit indicates decongestion after therapy or congestion intervention, or (e) said level of Pro-Adrenomedullin or its fragments is correlated with the assessment of the decision on hospital discharge, wherein a level elevated above a certain limit means that the individual would not be discharged and wherein a level below a certain limit means that the individual may be discharged, wherein said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRproADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CTproADM according to SEQ ID NO: 6.

[00158] 18. Use of at least one ligand in the manufacture of a reagent or a medicine or a kit for a) to diagnose congestion or to assess or monitor the extent of congestion in an individual or, b) to predict or determine or monitor the need for therapy or intervention or to predict or determine or monitor the success of a congestion therapy or intervention or to guide a therapy or intervention in an individual or, c) to predict residual decongestion or congestion after congestion therapy or intervention in an individual or, d) assess residual decongestion or congestion after Petition 870230018821, dated 06 / 03 / 2023, page 63 / 138 56 / 98 congestion therapy or intervention in an individual or, (e) to assess the decision regarding hospital discharge of an individual, wherein said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF, or wherein said individual has chronic heart failure with worsening signs / symptoms of chronic heart failure, wherein the ligand binds to a region within the amino acid sequence of Pro-Adrenomedullin or its fragments of at least 5 amino acids and can determine the level of immunoreactive analyte in a body fluid sample obtained from said individual, and Pro-Adrenomedullin or its fragments of at least 5 amino acids are used as an early surrogate marker for congestion; and wherein a) said level of immunoreactive analyte is correlated with the extent of congestion in said individual or diagnosis of congestion, where a level elevated above a certain limit is indicative of congestion or, b) said level of immunoreactive analyte is correlated with the success of a therapy or intervention in said individual, wherein a level below a certain threshold is predictive of therapy or intervention success, and wherein a level above a certain threshold is indicative of the need for therapy or intervention; c) said level of immunoreactive analyte is correlated with a prognosis of decongestion or residual congestion after congestion therapy or intervention, in Petition 870230018821, dated 06 / 03 / 2023, page 64 / 138 57 / 98 that a level elevated above a certain limit indicates residual congestion after therapy or congestion intervention, while a level below a certain limit indicates decongestion after therapy or, d) said immunoreactive analyte level is correlated with decongestion or residual congestion after therapy or congestion intervention, wherein a level elevated above a certain limit indicates residual congestion after therapy or congestion intervention, while a level below a certain limit indicates decongestion after therapy or congestion intervention, or (e) said immunoreactive analyte level is correlated with the assessment of the decision on hospital discharge where a level elevated above a certain limit means that the individual would not be discharged and where a level below a certain limit means that the individual can be discharged, where said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRproADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CTproADM according to SEQ ID NO: 6.

[00159] 19. Use in accordance with item 17 or 18, where said extent of congestion is expressed as a congestion score, in particular a clinical congestion score.

[00160] 20. The use in accordance with any of items 17 to 19, in which said individual is stratified. a) in groups of congestion degrees or, b) in non-responder and / or responding and / or bad Petition 870230018821, dated 06 / 03 / 2023, page 65 / 138 58 / 98 responder to congestion therapy or intervention or, c) in a decongested group or in a residual congestion group after congestion therapy or intervention.

[00161] 21. The use in accordance with any of items 17 to 20 in which said fragment may be selected from MR-proADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4.

[00162] 22. Use in accordance with any of items 17 to 21, wherein the ligand is selected from the group comprising an antibody, an antibody fragment or a non-Ig structure that binds to ProAdrenomedullin or its fragments of at least 5 amino acids.

[00163] 23. Use in accordance with any of the preceding items, wherein said limit is within a limit range which is a limit range for plasma ADM-NH2 according to SEQ ID NO: 4 between 50 and 100 pg / mL and for plasma MR-proADM between 0.5 and 1.5 nmol / L, and for plasma CT-proADM between 85 and 350 pmol / L.

[00164] 24. Use in accordance with any of items 17 to 23, wherein said determination of Pro-Adrenomedullin or its fragments of at least 5 amino acids is performed more than once in an individual.

[00165] 25. Use in accordance with any of items 17 to 24, where the sample is taken on admission to a hospital or on hospital discharge.

[00166] 26. Use in accordance with any of items 17 to 25, wherein said monitoring is performed in order to assess the response of said individual to the preventive and / or therapeutic measures taken.

[00167] 27. The use in accordance with any of items 17 to 26 in order to stratify said individuals into groups according to degree of congestion.

[00168] 28. The use in accordance with any of items 17 to 27 in which said level of Pro-Adrenomedullin or its fragments or said level Petition 870230018821, dated 06 / 03 / 2023, page 66 / 138 A level of 59 / 98 immunoreactive analyte is correlated with a risk of death or an adverse event in individuals with acute heart failure, where a level elevated above a certain threshold is predictive of an increased risk of death or adverse events.

[00169] 29. Use in accordance with any of items 17 to 28 wherein said level of Pro-Adrenomedullin or its fragments or said level of immunoreactive analyte is used for guidance of therapy or intervention, wherein therapy or intervention is indicated if said level of Pro-Adrenomedullin or fragments is above a certain limit and wherein therapy or intervention is not indicated if said level of Pro-Adrenomedullin or fragments is below a certain limit.

[00170] 30. Use in accordance with any of items 17 to 29 wherein said level of Pro-Adrenomedullin or its fragments or said level of immunoreactive analyte is used to determine the need for therapy or intervention.

[00171] 31. Use in accordance with any of items 17 to 30 where said intervention or therapy is selected from the group comprising the administration of diuretics, the administration of inotropes, the administration of vasodilators, ultrafiltration.

[00172] 32. A kit or reagent or medicine for a) to diagnose congestion or to assess or monitor the extent of congestion in an individual or, b) to predict or determine or monitor the need for therapy or intervention or to predict or determine or monitor the success of a congestion therapy or intervention or to guide a therapy or intervention in an individual or, c) to predict residual decongestion or congestion after congestion therapy or intervention in a Petition 870230018821, dated 06 / 03 / 2023, page 67 / 138 60 / 98 individual or, d) to assess residual decongestion or congestion after congestion therapy or intervention in an individual or, (e) to assess the decision regarding the hospital discharge of an individual, wherein said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF, or wherein said individual has chronic heart failure with worsening signs / symptoms of chronic heart failure, and wherein the kit or reagent or medication comprises a ligand that binds to Pro-Adrenomedullin or its fragments of at least 5 amino acids, and the ligand can determine the level of Pro-Adrenomedullin or its fragments of at least 5 amino acids in a sample of body fluid obtained from said individual; and wherein a) said level of Pro-Adrenomedullin or its fragments is correlated with the extent of congestion in said individual or diagnosis of congestion, where a level elevated above a certain threshold is indicative of congestion or, b) said level of Pro-Adrenomedullin or its fragments is correlated with the success of a therapy or intervention in said individual, wherein a level below a certain threshold is predictive of therapy or intervention success, and wherein a level above a certain threshold is indicative of the need for therapy or intervention; c) said level of Pro-Adrenomedullin or its fragments is correlated with a prognosis of Petition 870230018821, dated 06 / 03 / 2023, page 68 / 138 61 / 98 decongestion or residual congestion after congestion therapy or intervention, where a level above a certain limit indicates residual congestion after congestion therapy or intervention, while a level below a certain limit indicates decongestion after congestion therapy or intervention, or d) said level of Pro-Adrenomedullin or its fragments is correlated with decongestion or residual congestion after therapy or congestion intervention, wherein a level elevated above a certain threshold indicates residual congestion after therapy or congestion intervention, while a level below a certain threshold indicates decongestion after therapy or congestion intervention, or (e) said level of Pro-Adrenomedullin or its fragments is correlated with the assessment of the decision on hospital discharge, wherein a level elevated above a certain limit means that the individual would not be discharged and wherein a level below a certain limit means that the individual may be discharged, wherein said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRproADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CTproADM according to SEQ ID NO: 6.

[00173] 33. A kit or reagent or medicine for a) to diagnose congestion or to assess or monitor the Petition 870230018821, dated 06 / 03 / 2023, page 69 / 138 62 / 98 extent of congestion in an individual or, b) to predict or determine or monitor the need for therapy or intervention or to predict or determine or monitor the success of a congestion therapy or intervention or to guide a therapy or intervention in an individual or, c) to predict residual decongestion or congestion after congestion therapy or intervention in an individual or, d) to assess residual decongestion or congestion after congestion therapy or intervention in an individual or, (e) to assess the decision regarding hospital discharge of an individual, wherein said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF, or wherein said individual has chronic heart failure with worsening signs / symptoms of chronic heart failure, and wherein the kit or reagent or medication comprises at least one ligand that binds to a region within the amino acid sequence of ProAdrenomedullin or its fragments of at least 5 amino acids, and the ligand can determine the level of immunoreactive analyte in a sample of body fluid obtained from said individual; and wherein a) said level of immunoreactive analyte is correlated with the extent of congestion in said individual or diagnosis of congestion, where a level elevated above a certain limit is indicative of congestion or, b) said level of immunoreactive analyte is correlated Petition 870230018821, dated 06 / 03 / 2023, page 70 / 138 63 / 98 with the success of a therapy or intervention in said individual, where a level below a certain threshold is predictive of therapy or intervention success, and where a level above a certain threshold is indicative of a need for therapy or intervention, or c) said level of immunoreactive analyte is correlated with a prognosis of decongestion or residual congestion after congestion therapy or intervention, where a level elevated above a certain limit indicates residual congestion after congestion therapy or intervention, while a level below a certain limit indicates decongestion after congestion therapy or intervention, or (d) said level of immunoreactive analyte is correlated with decongestion or residual congestion after congestion therapy or intervention, where a level elevated above a certain limit indicates residual congestion after congestion therapy or intervention, while a level below a certain limit indicates decongestion after congestion therapy or intervention, or (e) said level of immunoreactive analyte is correlated with the assessment of the decision regarding hospital discharge, where a level elevated above a certain limit means that the individual would not be discharged and where a level below a certain limit means that the individual can be discharged, where said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MR Petition 870230018821, dated 06 / 03 / 2023, page 71 / 138 64 / 98 proADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CTproADM according to SEQ ID NO: 6.

[00174] 34. The kit or reagent or medicine according to item or 33, wherein said fragment may be selected from MR-proADM according to SEQ ID NO: 3 or ADM-NH2 according to SEQ ID NO: 4.

[00175] 35. The kit or reagent or medicament according to any of items 32 to 34, wherein the ligand is selected from the group comprising an antibody, an antibody fragment or a non-Ig structure that binds to Pro-Adrenomedullin or its fragments of at least 5 amino acids.

[00176] 36. The kit or reagent or medication according to any of items 32 to 35, wherein said limit is within a limit range which is a limit range for plasma ADM-NH2 according to SEQ ID NO: 4 between 50 and 100 pg / mL and for plasma MR-proADM between 0.5 and 1.5 nmol / L, and for plasma CT-proADM between 85 and 350 pmol / L. Description of the Figures Figure 1:

[00177] Figure 1 shows a typical bioADM dose / signal curve and a bioADM dose-signal curve in the presence of a 100 μg / mL NT-H antibody. Figure 2:

[00178] Serial bioADM measurement in patients with different degrees of congestion. Figure 3:

[00179] Illustration of the interaction between diuretic treatment on release and bioADM (admission). Kaplan-Meier plot of treatment with and without diuretics, separately for patients with bioADM. Petition 870230018821, dated 06 / 03 / 2023, page 72 / 138 65 / 98 below or above 70 pg / mL. Diuretics may be more beneficial in those with elevated bio-ADM on admission. FALSE 0: bio-ADM < 70 pg / mL, not treated with diuretics FALSE 1: bio-ADM < 70 pg / mL, treated with diuretics TRUE 0: bio-ADM > 70 pg / mL, not treated with diuretics TRUE 1: bio-ADM > 70 pg / mL, treated with diuretics Figure 4:

[00180] BioADM box plot for patients with significant congestion (CCS = 3) versus patients with no congestion or mild congestion (CCS < 3, p < 0.001). Figure 5:

[00181] BioADM box plot for patients with significant congestion (CCS = 3) versus patients with no congestion or mild congestion (CCS < 3, p < 0.01). Examples Example 1 Antibody Generation and Determination of their Affinity Constants

[00182] We developed mouse monoclonal antibodies that bind to the N-terminal (NT-ADM), mid-regional (MR-ADM) and C-terminal (CT-ADM) of bio-ADM and their affinity constants were determined (Table 1). Peptides for Immunization

[00183] The peptides were supplied by JPT Peptide Technologies GmbH (Berlin, Germany). The peptides were coupled to BSA using the Sulfo-SMCC crosslinking method. The crosslinking procedure was performed according to the manufacturer's instructions (Thermo Fisher / Pierce). Murine Antibody Generation

[00184] A Balb / c mouse was immunized with 100 μg of Petition 870230018821, dated 06 / 03 / 2023, page 73 / 138 66 / 98 conjugate of Peptide-BSA on days 0 and 14 (emulsified with 100 μL of complete Freund's adjuvant) and 50 μg on days 21 and 28 (with 100 μL of incomplete Freund's adjuvant). Three days before the fusion experiment was performed, the animal received 50 pg of the conjugate dissolved in 100 pL of saline solution, administered as an intraperitoneal and intravenous injection.

[00185] Splenocytes from immunized mice and SP2 / 0 myeloma cell line cells were fused with 1 mL of 50% polyethylene glycol for 30 s to 37°C. After washing, the cells were seeded in 96-pool cell culture plates. Hybrid clones were selected by culturing in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal calf serum and 11 HAT-Supplement). After two weeks, the HAT medium was replaced with HT medium for three passages, followed by return to normal cell culture medium.

[00186] Cell culture supernatants were subjected to primary screening for antigen-specific IgG antibodies three weeks after fusion. Microcultures that tested positive were transferred into 24-reservoir plates for propagation. After repeat testing, selected cultures were cloned and cloned again using the limiting dilution technique and isotypes were determined (Lane, 1985. J. Immunol. Meth. 81: 223-228; Ziegler et al. 1996. Horm. Metab. Res. 28: 11-15). Table 1: Antigen / Immunogen ADM Region Designation Affinity Constants Kd (M-1) YRQSMNNFQGLRSFGC 1-16 NT-ADM 1.6 x 109 Petition 870230018821, dated 06 / 03 / 2023, page 74 / 138 67 / 98 CTVQKLAHQIYQ 21-32 MR-ADM 2 x 109 CAPRSKISPQGY-NH2 C-42-52 CT-ADM 1.1 x 109 Production of Monoclonal Antibodies

[00187] Antibodies were produced using standard antibody production methods (Marx et al, 1997. Monoclonal Antibody Production, ATLA 25, 121) and purified using Protein A. Antibody purities were > 95% based on SDS gel electrophoresis analysis. Affinity Constants

[00188] To determine the affinity of the antibodies to Adrenomedullin, the binding kinetics of Adrenomedullin to the immobilized antibody were determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibodies was performed using an Fc anti-mouse antibody covalently coupled at high density to a CM5 sensor surface according to the manufacturer's instructions (mouse antibody capture kit; GE Healthcare). Marking Procedure (tracer)

[00189] 100 μg (100 μL) of antibody (1 mg / mL in PBS, pH 7.4) were mixed with 10 μL of NHS-acridinium ester (1 mg / mL in acetonitrile, InVent GmbH, Germany) (EP 0 353 971) and incubated for 20 min at room temperature. Labeled CT-H was purified by HPLC gel filtration on Bio-Sil® SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified labeled antibody was diluted in (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 5 g / L bovine serum albumin, pH 7.0). The final concentration was approximately 800 relative light units (RLU) of compound Petition 870230018821, dated 06 / 03 / 2023, p. 75 / 138 68 / 98 labeled (approximately 20 ng of labeled antibody) per 200 μL. Acridinium ester luminescence was measured using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG). Solid phase

[00190] Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with antibody (1.5 pg antibody / 0.3 mL of 100 mmol / L NaCl, 50 mmol / L TRIS / HCl, pH 7.8). After blocking with 5% bovine serum albumin, the tubes were washed with PBS, pH 7.4, and vacuum dried. Calibrators

[00191] Synthetic human ADM (hADM) (Bachem, Switzerland) was linearly diluted using 50 mM Tris / HCl, 250 mL 0.2% NaCl, 0.2% Triton X-100, 0.5% BSA, 20 tablets / L of Complete Protease Inhibitor Cocktail Tablets (Roche AG); pH 7.8. Calibrators were stored at -20°C before use. Example 2 Determining the antibody combination that produces high signal-to-noise ratios. ADM immunoassay

[00192] 50 pL of sample (or calibrator) was pipetted into coated tubes. After the addition of a second labeled antibody (200 pL), the tubes were incubated for two hours at room temperature. Unbound tracer was removed by washing 5 times (1 mL each time) with washing solution (PBS 20 mM, pH 7.4, Triton X-100 0.1%). Tube-bound chemiluminescence was measured using the LB 953 (Berthold Technologies GmbH & Co. KG).

[00193] All antibodies were used in an intercalated immunoassay as a coated tube and labeled antibody and combined in the following variations (see Table 2). Incubation was performed as described under the hADM immunoassay. The results are given in Petition 870230018821, dated 06 / 03 / 2023, page 76 / 138 69 / 98 ratio of specific signal (in 10 ng / mL of DM) / background signal (sample without DM). Table 2: Signal-to-noise ratio NT-ADM tracer MR-ADM tracer CT-ADM tracer NT-ADM / 195 241 MR-ADM 204 / 904 CT-ADM 260 871 /

[00194] Surprisingly, we observed the combination of MRADM and CT-ADM as a combination for the highest signal-to-noise ratio.

[00195] Subsequently, we used this antibody combination for other investigations to measure bio-ADM. We used anti-MR-ADM as the solid-phase antibody and anti-CT-ADM as the labeled antibody. A typical dose / signal curve is shown in Figure 1. The analytical sensitivity (average of 10 series, ADM-free sample + 2SD) of the assay was 2 pg of ADM / mL. Example 3 Stability of human adrenomedullin

[00196] Human ADM was diluted in human citrate plasma (n = 5, final concentration 10 ng ADM / mL) and incubated at 24°C. At selected times, aliquots were frozen at -20°C. Immediately after thawing, hADM samples were quantified using the hADM immunoassay described above.

[00197] Table 3 shows the stability of hADM in human plasma at 24°C. Petition 870230018821, dated 06 / 03 / 2023, page 77 / 138 70 / 98 Time (h) Average recovery of ADM (N=5) Relative loss of immunological reactivity Loss of immunological reactivity % / h 0 100 / / 2 99.2 0.8 0.4 4 96.4 3.6 0.8 8 88.2 11.8 1.5 Average: 0.9% / h

[00198] Surprisingly, using combinations of MR-ADM and CT-ADM antibodies in an intercalated immunoassay, the preanalytical stability of the analyte is high (only 0.9% / h mean loss of immunological reactivity). In contrast, using other assay methods, a plasma half-life of only 22 minutes has been reported (Hinson et al. 2000 Endocrine Reviews 21(2):138-167). Since the time to take the sample for routine hospital analysis is less than two hours, the ADM detection method used is suitable for routine diagnosis. It is noteworthy that any non-routine additives to the samples (such as aprotinin, (Ohta et al. 1999. Clin Chem 45 (2): 244-251)) are not necessary to achieve acceptable ADM immune reactivity stabilities. Example 4 Reproducibility of calibrator preparations

[00199] We observed a high variation in results when preparing calibrators for ADM assays (mean CV 8.5%, see Table 4). This may be due to the high adsorption of hADM on plastic and glass surfaces (Lewis et al. 1998. Clinical Chemistry 44 (3): 571-577). This Petition 870230018821, dated 06 / 03 / 2023, page 78 / 138 The 71 / 98 effect was only slightly reduced by the addition of detergents (up to 1% Triton X 100 or 1% Tween20), protein (up to 5% BSA), and high ionic strength (up to 1M NaCl) or combinations thereof. Surprisingly, if an excess of anti-ADM antibody (10 μg / mL) is added to the calibrator dilution buffer, the recovery and reproducibility of the ADM assay calibrator preparations was substantially improved for < 1% of the inter-preparation CV (Table 4).

[00200] Fortunately, the presence of terminal antibodies did not affect the bioADM signal generated by the combination of MR and C-terminal antibodies (Figure 1). Table 4: In the presence of NTADM antibody (10 pg / mL) Inter-preparation CV (%) Without antibody Inter-preparation CV (%) calibrator 100 ng / mL 3453 s / nr 0.9 2842 s / nr 2.8 10 ng / mL 1946 s / nr 0.8 1050 s / nr 7.9 1 ng / mL 179 s / nr 1.1 77 s / nr 14.8 Average: 0.93 Average: 8.5 Variation in the Interpreparation of Calibrators

[00201] ADM assay calibrators were prepared as described above with and without 10 pg / mL of NT-ADM antibody. Coefficients of variation are provided from 5 independent preparation operations. The calibrators were measured using the ADM assay described above (s / nr = signal-to-noise ratio). For all subsequent studies, we used a calibrator-based ADM assay prepared in the presence of 10 pg / mL of antibody. Petition 870230018821, dated 06 / 03 / 2023, p. 79 / 138 72 / 98 NT-ADM and 10 μg / mL of NT-ADM antibody as a supplement in the tracer buffer. Example 5 Sensitivity

[00202] The aim of the assay sensitivity was to fully cover the ADM concentration of healthy individuals. BioADM concentration in healthy individuals

[00203] Healthy individuals (n = 100, mean age 56 years) were measured using the bio-ADM assay. The mean value was 24.7 pg / mL, the lowest value was 1 pg / mL, and the 99 percent was 43 pg / mL. Since the sensitivity of the assay was 2 pg / mL, 100% of all healthy individuals were detectable using the described bioADM assay.

[00204] A fully automated commercial homogeneous time-resolved fluoroimmunoassay was used to measure MR-proADM in plasma (BHMS MR-proADM KRYPTOR; BHMS GmbH, Hennisdorf, Germany) (Caruhel et al. 2009. Clin Biochem. 42 (7-8):725-8). Example 6 (PROTECT) Study Population and Measurements

[00205] Details of this study have been published (Massie et al. 2010. N Engl J Med. 363:1419-1428.; Weatherley et al. 2010. J Card Fail. 16:25-35.; Voors et al. 2011. J Am Coll Cardiol. 57:1899-1907). In summary, 2033 patients with acute heart failure with impaired renal function (creatinine clearance estimated between 20 and 80 mL / min using the Cockcroft-Gault formula) were included and randomly assigned to rolophyllin or placebo. The PROTECT study protocol was approved by the ethics committee at each participating center, and written informed consent was obtained from all participants.

[00206] Bio-ADM was measured from plasma collected during baseline assessment in 1572 hospitalized AHF patients included. Petition 870230018821, dated 06 / 03 / 2023, page 80 / 138 73 / 98 in the PROTECT trial (all available baseline samples) using an immunoassay developed by Sphingotec GmbH (Hennigsdorf, Germany). PROTECT (meaning the Randomized, Placebo-Controlled Study of the Selective Adenosine Receptor Antagonist Rolophylline for Hospitalized Patients with Acute Decompensated Heart Failure and Volume Overload to Evaluate the Effect of Treatment on Congestion and Renal Function) was a multicenter, randomized, double-blind trial comparing rolophylline versus placebo in 2033 hospitalized patients for AHF. Study Results Clinical Congestion Score

[00207] As highlighted earlier, clinical surrogates have less than ideal predictive value for detecting congestion. In this analysis, we combined three of the strongest clinical surrogates for congestion (i.e., JVP, peripheral edema, and orthopnea) to improve accuracy and develop a composite clinical congestion score (CCS) using the scheme presented below: Parameter 0 1 2 3 Peripheral edema 0 Ankle Below the knee Above the knee Orthopnea 0 support point 1 support point 2 support points 3 support points JVP < 6 cm 6 to 10 cm > 10 cm -

[00208] The scores for each of these three parameters were then added together to obtain a composite congestion score that ranged from 0 to 8. A similar scheme was previously used by Ambrosy et al. (Ambrosy et al. 2013. European Heart Journal 34 (11): 835-843).

[00209] The following algorithm was then used to classify the severity of congestion: Petition 870230018821, dated 06 / 03 / 2023, page 81 / 138 74 / 98 CCS = 0, No clinical congestion CCS 1 to 3, Mild clinical congestion CCS 4 to 5, Moderate clinical congestion CCS > 6, Severe clinical congestion Decongestion Markers

[00210] Diuretic response: defined as weight loss by day 4 at a 40 mg dose of diuretic.

[00211] Hemoconcentration: coded as 0 (if there is a decline or no change in hemoglobin levels by day 4 compared to the reference value) or 1 (if there is an increase in hemoglobin levels by day 4 compared to the reference value).

[00212] Significant residual congestion: defined as a CCS > 2 based on JVP, orthopnea, and edema assessments by day 7. Statistical Analysis

[00213] Basic clinical features and biomarkers including bio-ADM were summarized by the severity of clinical congestion at the reference value (scheme shown above). The basic factors independently associated with the severity of clinical congestion at the reference value were determined using a multivariate logistic regression model (the CCS variable was recoded as a binary outcome with two levels; 0 = mild / moderate (CCS < 6) and 1 - severe (CCS > 6)).

[00214] The association between bioADM levels at baseline and diuretic response (a continuous variable) was assessed using linear regression analysis. For hemoconcentration and significant residual congestion outcomes, binary logistic regression analysis was performed. Multivariate models were used to assess the adjusted associations between bioADM levels and these outcomes. Results Petition 870230018821, dated 06 / 03 / 2023, page 82 / 138 75 / 98

[00215] Table 5 shows that bioADM concentrations increase with the severity of congestion. Table 5: Clinical variables and biomarkers at baseline by severity of clinical congestion at baseline Mild clinical congestion N = 212 Moderate clinical congestion N = 528 Severe clinical congestion N = 667 Trend P Male sex, % (N) 65.6 (139) 68.4 (361) 65.4 (436) 0.656 Age (years) 71.5 ± 11.7 71.1 ± 11.3 70.8 ± 10.7 0.427 BMI (Kg / m2) 26.5 ± 4.4 27.8 ± 5.3 29.9 ± 6.4 <0.001 LVEF (%) 32.1 ± 13.4 33.2 ± 13.4 32.2 ± 13.4 0.808 SBP (mmHg) 124.2 ± 17.9 125.2 ± 17.4 124.3 ± 17.3 0.773 BPD (mmHg) 72.9 ± 12.2 73.3 ± 11.4 74.1 ± 11.6 0.118 Heart Rate (bpm) 77.6 ± 13.6 79.1 ± 15.4 81 ± 15.9 0.002 Respiratory Rate (per min) 20 [18 - 22] 20 [18 - 24] 21 [18 - 24] 0.001 Orthopnea, % (N) None 17.9 (38) 4 (21) 0 (0) ref. One support point (10 cm) 36.8 (78) 14.8 (78) 2.5 (17) <0.001 Two support points (20 cm) 33.0 (70) 56.6 (299) 31.8 (212) <0.001 > 30 degrees 12.3 (26) 24.6 (130) 65.7 (438) <0.001 Pulmonary wheezing, % (N) < 1 / 3 25.5 (54) 33.5 (177) 28.9 (193) ref.1 / 3 - 2 / 3 57.1 (121) 51.5 (272) 48.9 (326) 0.333 > 2 / 3 17.5 (37) 15 (79) 22.2 (148) 0.101 Edema, % (N) 0 56.6 (120) 15.3 (81) 0 (0) ref. 1 + 29.2 (62) 33.1 (175) 5.7 (38) <0.001 2+ 13.2 (28) 43.2 (228) 47.2 (315) <0.001 3+ 0.9 (2) 8.3 (44) 47.1 (314) <0.001 JVP, % (N) < 6 cm 42.5 (90) 12.3 (65) 2.4 (16) ref. Petition 870230018821, dated 06 / 03 / 2023, p. 83 / 138 76 / 98 Mild clinical congestion N = 212 Moderate clinical congestion N = 528 Severe clinical congestion N = 667 Trend P 6 - 10 cm 52.4 (111) 62.3 (329) 34.9 (233) <0.001 > 10 cm 5.2 (11) 25.4 (134) 62.7 (418) <0.001 NYHA, % (N) I 0.9 (2) 1.5 (8) 0.7 (5) 0.784 II 24.1 (51) 15 (79) 14.4 (96) 0.779 III 50.5 (107) 50.4 (266) 47.4 (316) 0.668 IV 14.6 (31) 28.6 (151) Medical history COPD, % (N) 15.1 (32) 20.1 (106) 22 (146) 0.039 Stroke, % (N) 8 (17) 8.3 (44) 10.9 (73) 0.111 Peripheral vascular disease, % (N) 12.3 (26) 11.6 (61) 9.3 (62) 0.145 Hypertension, % (N) 78.8 (167) 77.7 (410) 82.6 (551) 0.075 Diabetes mellitus, % (N) 38.7 (82) 44.1 (233) 48.1 (321) 0.013 Hypercholesterolemia, % (N) 58 (123) 50 (264) 51.1 (340) 0.189 Myocardial infarction, % (N) 52.8 (112) 50.1 (264) 49.4 (328) 0.424 Angina, % (N) 20.8 (44) 20.7 (109) 24 (160) 0.187 Ischemic heart disease, % 68.9 (146) 70.4 (371) 71.4 (475) 0.468 (N) Atrial fibrillation, % (N) 44.3 (94) 53.0 (279) 59,8 (396) <0.001 past hospitalization for FH, % 41.5 (88) 47.2 (249) 53.4 (356) 0.001 (N) PCI, % (N) 34.0 (71) 26.5 (139) 23.3 (154) 0.003 CABG, % (N) 26.3 (55) 20.6 (108) 22.5 (149) 0.514 Pacemaker, % (N) 9.9 (21) 11 (58) 11.4 (76) 0.556 Medications during hospital admission ACEI / ARB, % (N) 78.3 (166) 75.7 (399) 75.6 (504) 0.492 Beta-blocker, % (N) 82.1 (174) 75.1 (396) 75.1 (501) 0.089 MRA, % (N) 41.5 (88) 44.8 (236) 47.5 (317) 0.110 Digoxin, % (N) 20.3 (43) 30.6 (161) 31.3 (209) 0.009, Petition 870230018821, dated 06 / 03 / 2023, p. 84 / 138 77 / 98 Mild clinical congestion N = 212 Moderate clinical congestion N = 528 Severe clinical congestion N = 667 Trend P Biomarkers Albumin (g / dL) 4.0 [3.7 - 4.3] 3.9 [3.6 - 4.2] 3.8 [3.5 - 4.1] <0.001 ALTERNATIVELY (U / L) 20 [14 - 32] 21 [15 - 32] 21 [15 - 30] 0.299 AST (U / L) 23 [17 - 31] 24 [19 - 33] 25 [20 - 33] 0.210 Bicarbonate (mEq / L) 23.6 ± 3.4 24.2 ± 3.6 24 ± 3.9 0.485 BUN (mg / dL) 29 [22 - 42] 28 [21 - 40] 31 [22 - 42] 0.057 Chloride (mEq / L) 102 [99 - 105] 101 [98 - 104] 101 [98 - 104] 0.085 Creatinine (mg / dL) 1.4 [1.1 - 1.8] 1.4 [1.1 - 1.8] 1.4 [1.1 - 1.8] 0.59 Total Cholesterol (mg / dL) 154.5 [122 - 183.5] 146.5 [123 - 173.8] 134 [110 - 164] <0.001 Glucose (mg / dL) 126 [104.5 - 155] 126 [101 - 168] 128 [103 - 162] 0.663 Hemoglobin (g / dL) 12.6 ± 1.8 12.5 ± 1.8 12.5 ± 1.9 0.716 Platelet count (*10A9 / L) 221 [180 - 278] 218 [170 - 268] 211 [168 - 260] 0.020 Potassium (mmol / L) 4.3 ± 0.6 4.3 ± 0.6 4.3 ± 0.6 0.723 RBC count (*10A9 / L) 4.2 ± 0.6 4.2 ± 0.6 4.2 ± 0.6 0.200 Sodium (mmol / L) 140 [137 - 142] 140 [138 - 142] 140 [137 - 142] 0.139 Triglycerides (mmol / L) 96 [66 - 132.5] 89 [65 - 130] 83 [64 - 112] 0.003 Uric acid (mg / dL) 8.5 ± 2.5 8.8 ± 2.6 9.1 ± 2.5 0.003 WBC (*10A9 / L) 7.9 [6.5 - 10] 7.3 [6.2 - 9.1] 7.3 [5.9 - 9] 0.003 Hematocrit (%) 39.5 ± 5.4 39.4 ± 5.6 40.1 ± 5.9 0.09 BNP (pg / mL) 406.8 [255.8 - 648.2] 437.4 [247.9 - 797.1] 487.9 [274.8 - 874] 0.002 cTnl (pg / mL) 12.0 [5.8 - 38.2] 9.8 [5.2 - 20.1] 10.8 [5.8 - 22.7] 0.046 bio-ADM (pg / mL) 30.0 [20.2 - 46.1] 37.4 [22.5 - 66.8] 55.4 [31.4 - 102.6] <0.001

[00216] Furthermore, it was demonstrated by regression logistics, Multivariate analysis shows that bio-ADM is an independent indicator of congestion severity and the strongest among all other available variables (Table 6). Petition 870230018821, dated 06 / 03 / 2023, page 85 / 138 78 / 98 Table 6: Basic factors independently associated with clinical congestion severity at baseline value in a multivariate logistic regression model (severe versus mild / moderate) Variable Odds Ratio [95% CI] P-value past hospitalization for FH 1.40 [1.11 - 1.75] 0.004 BMI 1.06 [1.04 - 1.08] <0.001 Serum albumin 0.56 [0.43 - 0.74] <0.001 bio-ADM, log 1.58 [1.38 - 1.82] <0.001

[00217] Area under the curve (AUC) of the entire model = 0.69, Individual AUCs: bio-ADM = 0.66, BMI = 0.61, serum albumin = 0.58, past hospitalization for HF = 0.54 Main message from tables 5 and 6 There are very few clinical variables or biomarkers at baseline associated with the severity of basic clinical congestion, and bio-ADM appears to be, by a large margin, the strongest.

[00218] In addition, it was analyzed whether bio-ADM would predict decongestion. Table 7 shows that bio-ADM is an independent indicator of significant residual congestion by day 7. Table 7: Association between bio-ADM levels and markers in the decongestion reference value. Decongestion marker Unadjusted Adjusted Estimate [95% CI] P-value Estimate [95% CI] P-value Significant residual congestion by day 7* 1.98 [1.70 - 2.32] <0.001 1.30 [1.10 - 1.60]** 0.009 Diuretic response by day 4 0.02 [-0.02 - 0.06] 0.350 - - Hemoconcentration by day 4 0.82 [0.71 - 0.94] 0.004 0.90 [0.78 - 1.04]# 0.160 defined as a composite congestion score > 2 by day 7 ** adjusted for variables at baseline including orthopnea, JVP, peripheral edema, history of PCI, pacemaker, use of ACEI / ARB, BMI, DBF, BUN, hematocrit, and BNP Petition 870230018821, dated 06 / 03 / 2023, page 86 / 138 79 / 98# adjusted for clinical congestion score at the reference value (NB: there is a -30% omission in the hemoconcentration data) Main message from table 7 Bio-ADM reference levels independently indicate significant residual congestion by day 7.

[0219] As expected for a marker of congestion, bio-ADM concentrations were higher the more diuretics were used for therapy (Tables 8 and 9). Table 8: Total IV diuretic dose through day 7 or release (if earlier) in terms of bio-ADM levels at reference value; PROTECT Tercile 1 Tercile 2 Tercile 3 trend P Total IV diuretic dose 200.0 280.0 400.0 <0.001 through day 7 or release [92.6 - [135.5 - [200 (if earlier), mg 352.8] 494.4] 882] Table 9: Unadjusted and adjusted association between bioADM levels at baseline and total IV diuretic dose through day 7 or release if earlier; linear regression analysis; PROTECT Not adjusted Adjusted* B (if) P value β (if) bio-ADM P value 3.4 (0.3) < 0.001 2.2 (0.3) < 0.001 * adjusted for age, systolic blood pressure, creatinine, blood urea nitrogen (BUN), albumin, sodium, prior hospitalization for FH, composite congestion score at baseline (CCS), and BNP; it should be noted that the association was more robust for bioADM, CCS at baseline, and renal function in this model.

[00220] In the same set of samples, MR-proADM was also Petition 870230018821, dated 06 / 03 / 2023, page 87 / 138 80 / 98 determined. The basic characteristics by tertiles of MR-proADM are shown in Table 10: Similar to bio-ADM, increasing levels of MR-proADM were associated with increased edema extent. Table 10: Basic characteristics by tertiles of MR-proAD Variable Tercile 1 Tercile 2 Tercile 3 Trend PN=516 N=515 N=516 MR-proADM (nmol / L) 0.24 [0.14 - 0.29] 0.55 [0.47 - 0.67] 1.25 [0.94 - 1.88] Sex 65.9 (340) 64.3 (331) 69.4 (358) 0.235 Age (years) 70.1 ± 11.1 71.3 ± 10.9 71.3 ± 11.4 0.073 BMI (Kg / m2) 28 ± 5.5 28.9 ± 5.9 29.5 ± 6.5 <0.001 LVEF( %) 32.9 ± 13.4 33.5 ± 12.3 31.3 ± 13.8 0.178 SBP (mmHg) 126.3 ± 17.5 125.9 ± 17.1 122.2 ± 17.4 <0.001 DBP (mmHg) 74.1 ± 12.1 74.3 ± 11.6 72.6 ± 11.7 0.033 Heart Rate (bpm) 78.7 ± 14.4 79.8 ± 15.4 81.2 ± 16.5 0.008 Respiratory Rate (min) 20 [18 - 24] 20 [18 - 24] 22 [18.8 - 24] 0.698 Orthopnea None 3.5 (18) 4.3 (22) 3.3 (17) ref. One point of support 12.8 (66) 10.5 (54) 13.2 (68) 0.819 Two points of support 43.4 (224) 43.3 (223) 34.5 (178) 0.619 >30 degrees 39.1 (202) 41.2 (212) 48.1 (248) 0.439 Wheezing None 8.3 (43) 7.6 (39) 11.2 (58) ref.<1 / 3 24.6 (127) 29.1 (150) 32.6 (168) 0.849 1 / 3 - 2 / 3 58.3 (301) 53 (273) 45.7 (236) 0.012 >2 / 3 8.5 (44) 10.3 (53) 10.3 (53) 0.622 Edema 0 16.3 (84) 14.8 (76) 12.2 (63) ref. 1 + 24.8 (128) 17.5 (90) 15.3 (79) 0.324 2+ 39.7 (205) 40 (206) 40.5 (209) 0.112 3+ 19.2 (99) 27.8 (143) 31.8 (164) <0.001 JVP <6 cm 11 (57) 9.3 (48) 12.6 (65) ref. 6 - 10 cm 45.3 (234) 42.3 (218) 40.3 (208) 0.22 >10 cm 34.7 (179) 37.7 (194) 37.4 (193) 0.755 NYHA I 1.7 (9) 0.8 (4) 0.6 (3) 0.024 II 17.4 (90) 17.5 (90) 13.8 (71) 0.062 III 50.6 (261) 49.3 (254) 44.8 (231) 0.07. Petition 870230018821, dated 06 / 03 / 2023, p. 88 / 138 81 / 98 IV 25.6 (132) 29.1 (150) 33.7 (174) 0.114 COPD 18.6 (96) 19.2 (99) 21.9 (113) 0.185 Stroke 8.7 (45) 8 (41) 11.2 (58) 0.164 Peripheral vascular disease 10.1 (52) 11.9 (61) 11.9 (61) 0.37 Hypertension 79.1 (408) 83.3 (429) 78.7 (406) 0.876 Diabetes mellitus 44.6 (230) 46 (237) 47.5 (245) 0.349 Hypercholesterolemia 53.8 (277) 52.6 (271) 46.9 (242) 0.027 Myocardial infarction 49.2 (252) 50.5 (260) 50.4 (260) 0.708 Angina 22.5 (116) 21.2 (109) 23.9 (123) 0.59 Ischemic heart disease 69.2 (355) 72.8 (375) 69.6 (359) 0.898 Atrial fibrillation 48 (246) 56.5 (290) 58.8 (302) 0.001 Past hospitalization for FH 44.8 (231) 48.9 (252) 53.3 (275) 0.006 PCI 27.5 (140) 25.6 (131) 24.9 (127) 0.344 CABG 21.4 (109) 21.9 (112) 23.2 (119) 0.472 Pacemaker 8 (41) 13.4 (69) 13 (67) 0.012 ACEI / ARB 77.7 (401) 78.6 (404) 71.5 (369) 0.02 Beta-blocker 79.1 (408) 74.5 (383) 72.5 (374) 0.014 MRA 44.6 (230) 45.9 (236) 45 (232) 0.901 Digoxin 28.7 (148) 30.7 (158) 27.3 (141) 0.631 Standard Lab Parameters Albumin (g / dL) 3.9 [3.6 - 4.2] 3.9 [3.6 - 4.2] 3.8 [3.5 - 4] <0.001 ALT (U / L) 21 [15 - 30] 20 [15 - 31] 21 [15 - 34] 0.003 AST (U / L) 24 [18 - 31] 25 [19 - 32.8] 26 [19 - 37] 0.003 Bicarbonate (mEq / L) 24.5 ± 3.6 24.4 ± 3.7 23.3 ± 3.8 <0.001 BUN (mg / dL) 25 [19 - 33] 29 [22 - 38.2] 38 [28 - 51] <0.001 Chloride (mEq / L) 101 [99 - 104] 101 [99 - 104] 101 [97 - 104] 0.023 Creatinine (mg / dL) 1.2 [1 - 1.5] 1.4 [1.1 - 1.7] 1.6 [1.3 - 2.1] <0.001 Total Cholesterol (mg / dL) 151 [126 - 181.2] 146 [119 - 174] 129 [107.5 - 159.5] <0.001 Glucose (mg / dL) 129.5 [104 - 171.5] 126 [103 - 164] 128 [101 - 155] 0.013 Hemoglobin (g / dL) 12.8 ± 1.9 12.6 ± 1.7 12.2 ± 1.9 <0.001 Platelet count (*10A9 / L) 227 [180 - 277] 214.5 [172 - 266.5] 207 [161.5 - 263] 0.011 Potassium (mmol / L) 4.3 ± 0.6 4.2 ± 0.6 4.3 ± 0.6 0.22 RBC count (*10A9 / L) 4.3 ± 0.6 4.2 ± 0.6 4.1 ± 0.6 <0.001 Sodium (mmol / L) 140 [138 - 142] 140 [138 - 143] 139 [136 - 142] <0,001 Triglycerides (mmol / L) 101 [71 - 138.5] 83 [63 - 118] 81 [61 - 107] <0.001 Uric acid (mg / dL) 8.3 ± 2.4 8.9 ± 2.4 9.7 ± 2.8 <0.001 WBC (*10A9 / L) 7.7 [6.3 - 9.2] 7.3 [6.1 - 9.1] 7.2 [5.9 - 9.3] 0.591 Hematocrit (%) 40.4 ± 5.8 40 ± 5.5 38.9 ± 5.9 <0.001 Biomarkers IL-6 (pg / mL) 9.1 [5.3 - 16.5] 10.8 [6.4 - 19.9] 14.3 [8.8 - 26] 0.037 Petition 870230018821, dated 06 / 03 / 2023, page 89 / 138 82 / 98 CRP (ng / mL) 12566.3 [6334.9 - 25975.8] 13306.5 [7168.3 - 27844.1] 16044.9 [8412.3 - 29966.3] 0.015 D-Dimer (ng / mL) 134 [90.6 - 301] 155.5 [90.6 - 340.8] 180.3 [90.6 - 374.7] 0.017 PIGR (ng / mL) 292.2 [197.5 - 500.4] 363 [263.5 - 531.6] 578.8 [371.4 - 999] <0.001 Pentraxin-3 (ng / mL) 3.4 [2.2 - 6] 3.9 [2.8 - 6] 5.5 [3.6 - 8.9] <0.001 GDF-15 (ng / mL) 3.6 [2.5 - 5.8] 4 [3 - 6.1] 6.3 [4.3 - 6.3] <0.001 RAGE (ng / mL) 4.7 [3.4 - 6.5] 4.9 [3.6 - 6.4] 5.4 [4 - 7.4] <0.001 TNF-R1a (ng / mL) 2.6 [1.7 - 4] 2.9 [2.2 - 3.9] 4.4 [3.2 - 5.9] <0.001 PCT (pg / mL) 14 [8 - 31] 20 [11 - 41] 35.5 [18 - 78] <0.001 Myeloperoxidase (ng / mL) 33.9 [17.7 - 78.5] 30.9 [18.7 - 60] 32.8 [17 - 66.2] 0.209 Syndecan-1 (ng / mL) 7.7 [6.4 - 9.5] 8.1 [6.9 - 9.4] 9.6 [8.1 - 11.6] <0.001 Periostin (ng / mL) 4 [2.3 - 6.8] 5.8 [3.5 - 8.8] 7.3 [4.3 - 11.2] <0.001 Galectin-3 (ng / mL) 32 [25.2 - 43.1] 34 [26.5 - 44.1] 42.7 [32.5 - 55.9] <0.001 Osteopontin (ng / mL) 98.3 [66.3 - 149.9] 106.4 [74.2 - 150.3] 133.8 [100.7 - 190.8] <0,001 ET1 (pg / mL) 5.7 [4.2 - 7.6] 6.7 [5.1 - 9] 7.9 [6.1 - 11] <0.001 BNP (pg / mL) 310.8 [189.8 - 548.6] 477.2 [262.6 - 783.3] 630.1 [341.3 - 1063] <0.001 NT-proCNP (pg / mL) 34 [22 - 48] 42 [31 - 58] 53 [38 - 74.2] <0.001 ST-2 (ng / mL) 1.8 [0.9 - 5.6] 2.8 [1 - 5.7] 6.1 [2.7 - 10.9] <0.001 VEGFR (ng / mL) 0.3 [0.2 - 0.4] 0.4 [0.2 - 0.5] 0.5 [0.3 - 0.8] <0.001 Angiogenin (ng / mL) 1940 [1229.1 - 2950.6] 1979.3 [1388.1 - 3093.5] 1675.8 [1178.7 - 2552.6] 0.007 Mesothelin (ng / mL) 81.6 [65.6 - 97] 83.6 [73.6 - 94.2] 94.5 [83.7 - 108.1] <0.001 Neuropilin SAND (ng / mL) 10.6 [6.6 - 15.8] 11.6 [8.1 - 16.8] 14.8 [10.8 - 20.4] <0.001 Troy (pg / mL) 72 [49 - 110] 84 [64 - 115] 114 [83 - 153] <0.001 Bio-ADM 28 [17.2 - 53.5] 42.9 [27.1 - 74.2] 70.9 [40.3 - 120.3] <0.001 LTBR (ng / mL) 0.3 [0.2 - 0.5] 0.4 [0.3 - 0.5] 0.5 [0.4 - 0.7] <0.001 ESAM (ng / mL) 59.2 [53.4 - 68] 60.2 [55.6 - 65.8] 66.3 [60.5 - 71.7] <0.001 KIM-1 (pg / mL) 273.4 [169.1 - 484.6] 296 [181.3 - 480.4] 316.8 [199.3 - 492.9] 0.485 NGAL (ng / mL) 64.8 [44.2 - 98.6] 81.8 [54.2 - 123] 107.2 [70.4 - 175.2] <0.001 PSAP-B (ng / mL) 34 [25.3 - 49.7] 36.9 [27.9 - 47.8] 45.4 [34.1 - 61.1] <0.001 WAP4C (ng / mL) 18.2 [8.7 - 38.8] 22.6 [13.9 - 38.5] 45.8 [26.9 - 67.8] <0.001 cTnI (pg / mL) 9.3 [5.1 - 19.5] 10.9 [5.6 - 21.2] 12.5 [6.3 - 27.2] 0.078 Example 7 (BIOSTAT)

[00221] Additional analyses were performed on the BIOSTAT Study (Biology Study to Tailored Treatment in Chronic Heart Failure). The study has been described in detail (WWW.BIOSTAT-CHF.EU; Voors et al. Petition 870230018821, dated 06 / 03 / 2023, pages 90 / 138 83 / 98 (2016. Eur J Heart Fail. Jun;18(6):716-26). The Biology Study to Tailored Treatment in Chronic Heart Failure (BIOSTAT-CHF) included 2516 patients with signs of worsening and / or symptoms of heart failure from 11 European countries who were considered to have sub-optimal medical treatment. A further 1738 patients from Scotland were included in a validation group. Overall, both patient groups were well matched. Most patients were hospitalized for acute heart failure, and the remainder presented with signs of worsening and / or symptoms of heart failure in outpatient clinics. Approximately half of the patients were in New York Heart Association class II, and 7% vs 34% of patients in the index group vs validation group had heart failure with preserved ejection fraction.According to the study design, all patients used diuretics, but due to the inclusion criteria for both groups, the patients were not on ideal evidence-based medical therapy. In the follow-up phase, uptitration to the doses recommended by guidance was encouraged. Study population

[00222] Patients with the following inclusion criteria: • age >18 years with symptoms of onset or worsening of heart failure, • having objective evidence of documented cardiac dysfunction, • left ventricular ejection fraction < 40% OR, • plasma concentrations of BNP and / or N-terminal pro-brain natriuretic peptide (NT-proBNP) > 400 pg / mL or > 2000 pg / mL, respectively, • being treated with oral or intravenous furosemide > 40 mg / day or equivalent at the time of inclusion, • not previously treated with therapies based on Petition 870230018821, dated 06 / 03 / 2023, page 91 / 138 84 / 98 evidence [angiotensin-converting enzyme (ACE) inhibitors / angiotensin receptor antagonists (ARBs) and beta-blockers] or who are receiving < 50% of the target doses of these medications at the time of inclusion, • be anticipated to be initiated or uptitrated with ACE inhibitors / ARBs and / or beta-blockers by the attending physician.

[00223] Patients were registered as impatient or from outpatient clinics. Approximately 2 / 3 were hospitalized and 1 / 3 were observed in the outpatient setting.

[00224] A subset of patients comprising all patient types included in the trial (n = 1806), for whom biomarker measurements at the reference value were available, was analyzed in the present invention.

[00225] Similar to the PROTECT study (example 6), in the BIOSTAT study, increasing levels of bio-ADM were also correlated with increased edema severity (table 11). Table 11: Clinical variables in reference value and standard biomarkers by severity of peripheral edema; BIOSTAT No Ankle Below the knee Above the knee Tendency PN = 734 N = 532 N = 408 N = 132 Age (years) 68 [59.7 - 76.2] 70.7 [62.6 - 78.6] 72.4 [64.2 - 78.8] 74.3 [64.6 - 80.4] <0.001 BMI (Kg / m2) 26.5 [23.9 - 29.4] 27.1 [23.9 - 30.4] 28.7 [24.8 - 33.8] 30.5 [26.2 - 34.9] <0.001 LVEF( %) 30 [25 - 35] 30 [25 - 37] 30 [23 - 38] 35 [25 - 43.8] 0.002 BPD (mmHg) 75.8 ± 12.9 74.2 ± 12.8 74.3 ± 14.6 72.5 ± 13 0.004 Heart Rate (bpm) 75 [65 - 85] 78.5 [68.2 - 90] 80 [70 - 92] 80 [70 - 92.2] <0.001 Orthopnea 21.5 (158) 40.4 (215) 50.7 (207) 61.4 (81) <0.001 Wheezing 5.6 (41) 10.7 (57) 16.4 (67) 28.8 (38) <0.001 >1 / 3 lung fields Elevated JVP 11.2 (82) 29.7 (158) 38 (155) 47 (62) <0.001 Petition 870230018821, dated 06 / 03 / 2023, page 92 / 138 85 / 98 NYHA I 2.7 (20) 0.8 (4) 1 (4) 15.2 (20) ref. II 48.2 (354) 31 (165) 14.7 (60) 57.6 (76) 0.402 III 39.2 (288) 53.2 (283) 62.7 (256) 21.2 (28) <0.001 IV 8.2 (60) 13.5 (72) 18.1 (74) 6.1 (NA) <0.001 Hepatomegaly 8 (59) 16.2 (86) 22.3 (91) 27.3 (36) <0.001 Anamnesis Diabetes mellitus 27.1 (199) 32.7 (174) 43.6 (178) 36.4 (48) <0.001 Atrial fibrillation 38.6 (283) 49.1 (261) 56.4 (230) 55.3 (73) <0.001 ACEI / ARB 73.8 (542) 74.6 (397) 66.7 (272) 57.6 (76) <0.001 Beta-blocker 86.8 (637) 83.1 (442) 78.2 (319) 75 (99) <0.001 Biomarkers ALBUMIN1 (g / L) 33 [28 - 39] 32 [27 - 37.5] 31 [26 - 35] 30 [24.2 - 33.8] <0.001 AST (U / L) 24 [19 - 32] 26 [19 - 36] 28 [21 - 37] 29 [24 - 40.8] 0.003 Gamma-GT (U / L) 39.7 [24 - 79.5] 61 [30 - 107.8] 69 [40 - 142.3] 98.5 [55 - 174.5] <0.001 BUN (mmol / L) 10.1 [7.1 - 16.5] 12.0 [7.8 - 19.3] 11.9 [8.3 - 19.9] 11.3 [7.4 - 18.4] 0.001 Total cholesterol 4.4 [3.7 - 5.2] 4 [3.3 - 4.9] 3.8 [3.1 - 4.5] 3.1 [2.8 - 3.7] <0.001 (mmol / L) Hemoglobin 13.5 ± 1.8 13 ± 1.9 13.1 ± 1.9 12.4 ± 2.1 <0.001 (g / dL) Triglycerides 1.3 [1 - 1.8] 1.2 [0.9 - 1.5] 1.1 [0.9 - 1.5] 0.9 [0.8 - 1.2] <0.001 (mmol / L) Hematocrit (%) 40.6 ± 5.1 39.7 ± 5.4 39.8 ± 5.6 38.3 ± 5.8 <0.001 BNP (pg / mL) 489.1 [225 - 812] 663.8 [322 - 1192] 792.5 [487 - 1471] 759.0 [557 - 2023] 0.02 bio-ADM (pg / mL) 26.1 [19.2 - 37.8] 36.7 [24.6 - 54.4] 52.8 [34.4 - 86.7] 82.8 [49.4 - 141.2] <0.001,

[00226] In the same set of samples, MR-proADM was determined. The basic characteristics by tertiles of MR-proADM are shown in Table 12: Similar to bio-ADM, increasing levels of MR-proADM were associated with increased edema extent. Table 12: Basic characteristics by tertiles of MR-proADM Variable Tercile 1 Tercile 2 Tercile 3 Trend PN = 704 703 703 MR-proADM (nmol / L) 0.3 [0.2 - 0.3] 0.5 [0.4 - 0.6] 1 [0.8 - 1.5] <0.001 Male sex 74.6 (525) 72.1 (507) 73 (513) 0.498 Age (years) 65.3 [57 - 73.8] 71.2 [62.9 - 78.4] 74.3 [65.2 - 80.4] <0.001 BMI (Kg / m2) 26.8 [23.9 - 30.4] 27.3 [24.2 - 30.9] 27.1 [24.1 - 30.7] 0.051 Petition 870230018821, dated 06 / 03 / 2023, page 93 / 138 86 / 98 LVEF (%) 30 [25 - 35] 30 [25 - 37.2] 30 [25 - 38] 0.297 SBP (mmHg) 126.3 ± 20.8 125.8 ± 23.4 121.2 ± 21.1 <0.001 DBP (mmHg) 76.4 ± 13.3 75.2 ± 13.3 72.3 ± 13 <0.001 Heart Rate (bpm) 75 [65 - 86] 77 [68 - 90] 77 [67 - 90] 0.015 Orthopnea present 23 (162) 35.8 (252) 45.1 (317) <0.001 Wheezing >1 / 3 lung fields 6.4 (45) 11.9 (84) 12.9 (91) 0.074 Edema 0 45 (317) 32.9 (231) 23.2 (163) ref. 1+ 20.5 (144) 26.3 (185) 26.5 (186) <0.001 2+ 11.9 (84) 17.2 (121) 27.0 (190) <0.001 3+ 2.1 (15) 4.8 (34) 11.0 (77) <0.001 JVP High 14.9 (105) 20.9 (147) 32.4 (228) <0.001 NYHA I 2.7 (19) 2.6 (18) 1 (7) ref.II 46 (324) 37.7 (265) 21.5 (151) 0.751 III 40.1 (282) 48.1 (338) 55.3 (389) 0.002 IV 7.8 (55) 9.7 (68) 18.8 (132) <0.001 Hepatomegaly 9.7 (68) 12.8 (90) 19.8 (139) <0.001 S3 9.7 (68) 9.7 (68) 9.5 (67) 0.935 Medical history COPD 15.1 (106) 17.4 (122) 18.9 (133) 0.054 Stroke 7.1 (50) 10 (70) 11.5 (81) 0.005 Peripheral vascular disease 8.5 (60) 12.5 (88) 12.5 (88) 0.017 Hypertension 60.8 (428) 64.3 (452) 61.3 (431) 0.842 Diabetes mellitus 25.6 (180) 35.3 (248) 36.7 (258) <0.001 Myocardial infarction 29.7 (209) 39.5 (278) 42.1 (296) <0.001 Atrial fibrillation 37.1 (261) 46.4 (326) 53.5 (376) <0.001 Past hospitalization for FH 27.8 (196) 30.4 (214) 34.3 (241) 0.009. Petition 870230018821, dated 06 / 03 / 2023, p. 94 / 138 87 / 98 PCI 17 (120) 21.2 (149) 23.6 (166) 0.002 CABG 12.2 (86) 17.8 (125) 21.8 (153) <0.001 ACEI / ARB 75.7 (533) 73.1 (51.6) (40.4) <0.67 Beta - blocker 84.9 (598) 83.5 (587) 80.1 (563) 0.016 Standard lab parameters Albumin (g / L) 34 [29 - 39] 33 [27 - 37] 30 [24 - 36] <0.0 [0.01 U / L) 25 [17 - 39] 23 [15 - 35] 0.452 AST (U / L) 24 [20 - 33] 25.9 [18 - 35] 27 [20 - 36] 0.087 Gamma-GT (U / L) 40.8 [25 - 84] [25 - 65 - 69] 132.4] <0.001 Alkaline phosphatase (ug / L) 80 [63 - 114.8] 83 [65 - 111] 91 [69 - 123.5] 0.087 BUN (mmol / L) 8.9 [6.4 - 13.3] 11 [6.4 - 195 - 15 25] <0.001 Creatinine (mg / dL) 89.3 [75.8 - 106.1] 100 [84.6 - 123.4] 124.6 [98 - 167] 0.61 Total cholesterol (mmol / L) 4.5 [3.8 - 5, 4, 4.3 - 4] 3.6 [3 - 4.5] <0.001 Glucose (mg / dL) 6 [5.3 - 7.5] 6.4 [5.4 - 8.5] 6.5 [5.4 - 7.9] 0.22 Hemoglobin (g / dL) 13.7 ± 1.7 13.3.18 ± 1.29 <0.001 Platelet count (*10A9 / L) 221 [183 - 264] 220 [173 - 263] 208 [161 - 258] 0.001 Potassium (mmol / L 4.3 [4 - 4,6] 4.2 [3.9 - 4.5] 4.2 [3.8 - 4.6] 0.003 RBC Count (*10A12 / L) 4.6 [4.2 - 5] 4.5 [4.1 - 4.9] 4.2 [3.8 - 4.7] <0.001 Sodium (mmol / L) 140 [138 - 142] 140 [137 - 142] 139 [136 - 141] <0.001 Triglycerides (mmol / L) 1.3 [1 - 1.7] 1.3 [0.9 - 1.8] 1.1 [0.9 - 1.5] <0.001 WBC (*10A9 / L) 7.7 [6.6 - 9.2] 8 [6.5 - 9.8] 7.7 [6.3 - 9.7] 0.529 Hematocrits (%) 41.3 ± 4.9 40.2 ± 5.2 38.2 ± 5.4 <0.001 IL-6 biomarkers (pg / mL) 3.7 [2 - 6.8] 5.1 [2.8 - 8.9] 8.2 [4.5 - 16.5] <0.001 CRP (ng / mL) 11872.9 [4810.7 - 25356.7] 12266.6 [5506.4 - 25955.3] 16350.1 [7718.9 - 29386.1] 0.004 D-Dimer (ng / mL) 101.9 [101.9 - 101.9] 101.9 [101.9 - 146.5] 101.9 [101.9 - 194.3] 0.032 PIGR (ng / mL) 71.5 [44 - 106.5] 123.6 [89.8 - 177.3] 216.3 [153.8 - 311.2] <0.001, Petition 870230018821, dated 06 / 03 / 2023, page 95 / 138 88 / 98 Pentraxin-3 (ng / mL) 1.3 [0.8 - 2.1] 2 [1.4 - 3.1] 3.3 [2.1 - 5.2] <0.001 GDF-15 (ng / mL) 2.6 [2.2 - 3.1] 3.4 [3 - 4] 4.4 [3.8 - 5.3] <0.001 RAGE (ng / mL) 2 [1.4 - 2.7] 2.9 [2.1 - 4] 3.8 [2.8 - 5] <0.001 TNF-R1a (ng / mL) 0.6 [0.3 - 0.8] 1 [0.8 - 1.4] 1.9 [1.3 - 2.7] <0.001 PCT (pg / mL) 6.2 [3.7 - 16.1] 16 [8.2 - 31.6] 36.6 [18.9 - 82.2] <0.001 Myeloperoxidase (ng / mL) 24.3 [20.6 - 30.2] 28.4 [24.1 - 34.8] 31.1 [26.6 - 38.9] <0.001 Syndecan-1 (ng / mL) 1.2 [0.6 - 2.1] 2.2 [1.4 - 3.5] 3.9 [2.5 - 6.1] <0.001 Periostin (ng / mL) 3.9 [2.3 - 6.5] 6.2 [4 - 9.5] 9.2 [5.9 - 14.9] <0.001 Galectin-3 (ng / mL) 17.6 [13.2 - 24.7] 20.3 [15.4 - 27] 26,8 [19,1 - 37,7] <0,001 Osteopontin (ng / mL) 176,3 [147,2 - 206,4] 215,4 [187,2 - 247] 260,2 [227,7 - 298,8] <0,001 ET1 (pg / mL) 4,4 [3,5 - 5,9] 5,3 [4 - 6,7] 6,6 [5,1 - 9,1] <0,001 BNP (pg / mL) 515 [219,9 - 957,5] 598,3 [260,7 - 1162] 1032 [639 - 1641,5] 0,004 NT-proCNP (pg / mL) 5,2 [5,2 - 5,2] 5.3 [5.2 - 11.2] 14.7 [6.2 - 27.4] <0.001 ST-2 (ng / mL) 4.2 [2.3 - 9,1] 8.3 [4.5 - 15.6] 20.4 [10.1 - 36.1] <0.001 VEGFR (ng / mL) 0.1 [0.1 - 0.1] 0.1 [0.1 - 0.2] 0.2 [0.1 - 0.4] <0.001 Angiogenin (ng / mL) 6202.1 [4115.9 - 9654.5] 4484.4 [3193 - 6378.8] 3672 [2629 - 5151.6] <0.001 Mesothelin (ng / mL) 47.7 [42.4 - 53.1] 53.7 [48.7 - 59.5] 60.4 [54.2 - 68.8] <0.001 Neuropilin SAND (ng / mL) 17 [13.5 - 20.8] 21.3 [17.5 - 25.5] 26.6 [21.4 - 31.6] <0.001 Troy (pg / mL) 0.1 [0.1 - 0.2] 0.3 [0.2 - 0.4] 0.4 [0.3 - 0.7] <0.001 LTBR (ng / mL) 0.1 [0.1 - 0.1] 0.2 [0.1 - 0.2] 0.2 [0.2 - 0.3] <0.001 ESAM (ng / mL) 57.1 [51.6 - 62.2] 64.2 [59.2 - 69.1] 71 [65.8 - 78] <0.001 NGAL (ng / mL) 45.6 [30.7 - 72.4] 59 [37.5 - 87.6] 82.7 [51 - 135.4] <0.001 PSAP-B (ng / mL) 25.5 [17.2 - 33] 31.6 [24.4 - 37.4] 37 [29.4 - 43.1] <0.001 WAP4C (ng / mL) 0.7 [0.5 - 1.2] 1.4 [0.9 - 2.3] 3.5 [2 - 6.2] <0.001 cTnI (pg / mL) 9.6 [5 - 23.5] 12.6 [7.2 - 27] 16.5 [9.5 - 35.9] 0.098, Example 8 (Analysis of subgroup PROTECT)

[00227] Table 13 shows data from the PROTECT test that Petition 870230018821, dated 06 / 03 / 2023, page 96 / 138 89 / 98 association between bio-ADM levels and clinical congestion by left ventricular ejection fraction (LVEF) status. Since classification based on ejection fraction is the most relevant phenotyping currently used in heart failure, the analysis was performed on subgroups of HFrEF vs HFpEF. The association between bio-ADM levels and clinical congestion score is comparable in both subgroups. Table 13: Association between biol-ADM levels at reference value (log-transformed) and clinical congestion score by ejection fraction status in PROTECT Unadjusted OR (95% CI) Adjusted OR (95% CI) HFrEF HFpEF P* Interaction HFrEF HFpEF P* Interaction Clinical congestion at baseline 1.85 [1.50 - 2.30] 1.73 [1.10 - 2.86] 0.670 1.55 [1.25 - 1.95] 1.83 [1.08 - 3.33] 0.540 Significant residual congestion by day 7 2.91 [2.18 - 3.96] 2.86 [1.60 - 5.68] 0.450 1.90 [1.34 - 2.77] 2.10 [0.60 - 8.74] 0.802 = adjusted for BMI, serum albumin, and past hospitalization for HF for outcome 1 = adjusted for variables at baseline including orthopnea, JVP, peripheral edema, PCI history, pacemaker, ACEI / ARB use, diastolic blood pressure, blood urea nitrogen, hematocrit, and BNP for outcome 2 * interaction tested between log-transformed bio-ADM and left ventricular ejection fraction (LVEF), which was included as a continuous variable

[00228] Heart failure with reduced ejection fraction (HFrEF) was defined as LVEF < 40% and heart failure with preserved ejection fraction (HFpEF) as LVEF > 50%.

[00229] LVEF data was only available in 763 Petition 870230018821, dated 06 / 03 / 2023, page 97 / 138 90 / 98 patients (excluding the 1572 patients with bio-ADM measurements within the reference range available); of these, 545 had HFrEF while 102 had HFpEF. Example 9 Serial measurements (monitoring) of bio-ADM in patients with congestion (PROTECT study)

[00230] Bio-ADM was measured from plasma collected during follow-up assessment in hospitalized AHF patients included in the POTECT trial (all samples available on day 2 and day 7) to monitor bio-ADM levels in relation to congestion status. Mean bio-ADM levels for all patients at different time points are summarized in Table 14. Bio-ADM levels were analyzed over time through treatment success on day 7 (defined based on congestion status by day 7 using the composite congestion score [CCS] as explained above).

[00231] Figure 2 shows that patients with significant residual congestion by day 7 have high bio-ADM levels at the reference value, and these high levels are sustained after the course of 7 days of therapy, while declining levels compared to the reference value are observed by day 7 for patients with very little or no congestion by day 7. Table 14: Summary of bio-ADM levels for serial measurements of Bio-ADM levels in pg / mL (mean [interquartile range]) Day 1 (N = 1562) Day 2 (N = 1466) Day 7 (N = 1454) 44.1 [25.9 - 82.7] 43.9 [25.8 - 77.3] 34.3 [21.6 - 61.8] Example 10

[00232] Comparative analysis of the predictive value of bio-ADM and MRproADM for clinically assessed congestion Study PROTECT

[00233] In table 15, a comparative analysis of bio-ADM and MRPetition 870230018821, dated 06 / 03 / 2023, page 98 / 138 The 91 / 98 proADM score in terms of prognostic value for clinical congestion severity at baseline and the presence of significant residual congestion by day 7 in the PROTECT trial is presented. Clinical congestion was assessed using the composite congestion score (CCS) as previously described. Clinical congestion severity at baseline was classified as mild / moderate if the CCS was > 6 and severe if the CCS was > 6. Then, unadjusted and adjusted associations between bio-ADM levels, MR-proADM, and clinical congestion severity at baseline were evaluated using single-variable and multivariable binary logistic regression models.A basic multivariate model including body mass index, serum albumin, total cholesterol, BNP, history of atrial fibrillation, and past hospitalization for FH was identified for multivariate analysis after performing a backward selection on a model that included single-variable variables associated with clinical congestion severity at the reference value at a significance level of 20.0%. The area under the curve (AUC) was calculated for bio-ADM and MRproADM to quantify and compare the discriminatory value of the two biomarkers between mild / moderate and severe clinical congestion.

[00234] For residual congestion, the CCS assessed on day 7 was used. Significant residual congestion was considered present if the CCS on day 7 was > 3. Again, unadjusted and adjusted associations between bio-ADM levels, MR-proADM, and the presence of significant residual congestion on day 7 were assessed in single-variable and multivariate binary logistic regression models. A basic model including orthopnea, jugular venous pressure, peripheral edema, history of percutaneous intervention, pacemaker, use of ACEI / ARB, BMI, diastolic blood pressure, blood urea nitrogen, hematocrit, and BNP was identified for multivariate analysis after performing a backward selection on a model that included Petition 870230018821, dated 06 / 03 / 2023, pp. 99 / 138 92 / 98 single-variable variables were associated with the presence of significant residual congestion on day 7 at a significance level of 20.0%. The AUC was calculated to quantify and compare the discriminatory value of the biomarkers with respect to the presence of significant residual congestion. Table 15: Comparative analysis of bio-ADM and MR-proADM levels at baseline in the prognosis of congestion severity and in the presence of significant residual congestion by day 7 in PROTECT. Severity of clinically assessed congestion at reference value Biomarker Unadjusted Adjusted OR [95% CI] P-value AUC [95% CI] OR [95% CI] P-value bio-ADM (Ln-transformed) 1.76 [1.56 - 1.99] <0.001 0.66 [0.63 - 0.68] 1.44 [1.25 - 1.65] <0.001 MR-proADM 1.27 [1.14 - 1.41] <0.001 0.57 [0.54 - 0.60] 1.06 [0.93 - 1.20] 0.363 Presence of significant residual congestion by day 7 Unadjusted Adjusted OR [95% CI] P-value AUC [95% CI] OR [95% CI] P-value bio-ADM (Ln-transformed) 1.87 [1.62 - 2.17] <0.001 0.66 [0.63 - 0.70] 1.28 [1.06 - 1.54] 0.009 MR-proADM 1.36 [1.20 - 1.54] <0.001 0.59 [0.55 - 0.62] 0.92 [0.76 - 1.10] 0.362

[00235] Odds ratios should be interpreted by standard deviation of bio-ADM Ln-transformed and MR-proADM (untransformed) BIOSTAT study

[00236] Table 16 shows the association between bio-ADM and MRproADM levels and the severity of clinical congestion at the BIOSTAT reference value. Clinical congestion was assessed using a slightly different CCS (compared to that used in PROTECT), but a similar statistical approach was employed. Petition 870230018821, dated 06 / 03 / 2023, pages 100 / 138 93 / 98 was used for the analysis. CCS was calculated using edema, orthopnea, and jugular venous pressure, but the score only ranged from 0 to 5, as shown in the following table: Parameter 0 1 2 3 Peripheral edema 0 Ankle Below the knee Above the knee Orthopnea Absent Present - - JVP Not elevated Elevated - -

[00237] The severity of congestion at the reference value was defined as none / mild if the CCS was < 2 (N = 709) and significant if the CCS was > 2 (N = 635). A binary logistic regression model was used to assess unadjusted and adjusted associations. The same basic model identified in PROTECT (including BMI, serum albumin, total cholesterol, BNP, history of atrial fibrillation, and past hospitalization for FH) was used for multivariate adjustment. Table 16: Comparative analysis of bio-ADM and MR-proADM levels at the reference value in the prognosis of congestion severity at the reference value in BIOSTAT Severity of clinically assessed congestion at reference value Biomarker Unadjusted Adjusted OR [95% CI] P-value AUC [95% CI] OR [95% CI] P-value bio-ADM, (Lntransformed) 3.17 [2.72 - 3.73] <0.001 0.76 [0.73 - 0.78] 2.20 [1.78 - 2.75] <0.001 MR-proADM 2.62 [2.18 - 3.17] <0.001 0.69 [0.67 - 0.72] 1.82 [1.42 - 2.38] <0.001 Example 11 Study of GREAT Study Population

[00238] Three groups of unselected AHF patients (n = 1075) who present with acute dyspnea in the emergency department. Petition 870230018821, dated 06 / 03 / 2023, pages 101 / 138 94 / 98 of the participating university hospitals in three countries (United Kingdom, France, and Switzerland) were recruited. AHF was defined according to the European Society of Cardiology guidelines as progressive worsening or new onset of shortness of breath along with clinical signs of pulmonary or peripheral edema and elevated jugular venous pressure requiring intensification of diuretic and / or vasodilator therapy. Inclusion was independent of renal function, although patients with end-stage renal failure on established renal replacement therapy were excluded. These studies comply with the Declaration of Helsinki and ethical approvals granted from the respective research ethics committees. All patients provided written informed consent. Plasma Sampling

[00239] After signed informed consent, venous blood was drawn from supine patients and collected in pre-chilled tubes containing EDTA as an anticoagulant. The interval for obtaining this admission sample was up to 4 hours (Paris), 1 hour (Basel), and 12 hours (Leicester). Plasma was stored at -80°C until analysis in a single batch. Results

[00240] Patient characteristics are summarized in Table 17, and biomarker distribution is summarized in Table 18. During the first year of follow-up, 299 died. One of the groups (Paris) had no data due to rehospitalization and was excluded for this final stage. The remaining centers had follow-up data for n = 861 patients, of whom 345 died or were rehospitalized for heart failure (HF) within one year.

[00241] Bio-ADM was associated with 1-year mortality (p < 0.0001) and 1-year mortality or re-hospitalization for HF (p < Petition 870230018821, dated 06 / 03 / 2023, pages 102 / 138 95 / 98 0.0001), both for a single variable and for multiple variables (both p < 0.0001). Notably, there was an interaction between bio-ADM and diuretic treatment given at release (p = 0.0001, Tables 19 and 20 and Figure 3). The standardized HR (standardized to one standard deviation (SD) of bio-ADM transformed by logw) was 1.7 (p < 0.0001) for bio-ADM and 0.61 (p < 0.0001) for the interaction term with diuretic treatment. Admission Characteristics Number 1075 Male 643 (59.8%) Previous FH 361 (33.5%) Previous IHD 318 (29.6%) Previous Hypertension 597 (55.5%) Diabetes 339 (31.5%) Urea (mM) 8.8 [2.3 - 46.6] eGFR mL / min / 1.73m2 53.3 [5 - 111] Table 17: Characteristics of patients included in the analysis. NT-proBNP bio-ADM Admission 2165 [0.3 - 15902] 49.6 [10.5 - 1772.8] Release 1617 [0.3 - 16233] 44.5 [9.5 - 734.2] Table 18: Summary of biomarker distribution for patients included in the analysis at admission and discharge. χ2 df P Bio-ADM (Factor + higher-order factors 87.71 2 <0.0001) Total interactions 13.08 1 0.0003 Diuretic Effect (Factor + higher-order factors 96.29 2 <0.0001) Total interactions 13.08 1 0.0003 Bio-ADM x Diuretic Effect (Factor + higher-order factors 13.08 1 0.0003) Total 195.65 3 <0.0001 Table 19: Results of multivariate Cox regression analysis for end-stage mortality one year after admission, including the interaction term for diuretic treatment during release and bio-ADM. Petition 870230018821, dated 06 / 03 / 2023, pages 103 / 138 96 / 98 z2 df P Bio-ADM (Factor + higher-order factors) 60.19 2 <0.0001 Total interactions 15.69 1 0.0001 Diuretic Effect (Factor + higher-order factors) 82.86 2 <0.0001 Total interactions 15.69 1 0.0001 Bio-ADM x Diuretic Effect (Factor + higher-order factors) 15.69 1 0.0001 Total 142.37 3 <0.0001 Table 20: Results of multivariate Cox regression analysis for end-stage death or rehospitalization for heart failure (HF) one year after admission, including the interaction term for diuretic treatment on release and bio-ADM.

[00242] Bio-ADM was also associated with clinical congestion. Data were available from the Leicester and Paris centers (n = 1107). The clinical congestion score combines the presence of peripheral edema, orthopnea, and jugular venous pressure > 6 cm (each adding one point to the score, see Table 21). Bio-ADM was elevated in patients with a clinical congestion score (CCS) of 3 (n = 284, P < 0.001, Figure 4). Parameter 0 1 Peripheral edema no yes Orthopnea no yes Jugular venous pressure < 6 cm > 6 cm Table 21: Definition of the clinical congestion score (CCS). CCS = 0 CCS = 1 to 2 CCS = 3 Example 12: no congestion, mild congestion, significant congestion Petition 870230018821, dated 06 / 03 / 2023, pp. 104 / 138 97 / 98 DiSomma Study Study Population

[00243] This was a prospective observational experiment conducted in an intensive care unit. We enrolled patients admitted to AHF from the emergency department (ED) of Sant'Andrea Hospital in Rome. Clinical and laboratory data, including Clinical Congestion Score (CCS: peripheral edema, jugular vein distension, and orthopnea), diuretic treatment, and bio-ADM values, were collected upon arrival, and patients were followed until hospital discharge. The study was approved by the ethics committee of Sant'Andrea Hospital in Rome. All patients provided written informed consent. Plasma Sampling

[00244] Plasma samples for ADM measurement were obtained on admission. Results

[00245] 209 patients with a final diagnosis of AHF were recruited. Clinical congestion score was available in 168 patients. Patient characteristics are summarized in Table 17. 22.6%, 38.1%, 21.4%, and 17.9% of patients presented a CCS of 0, 1, 2, and 3, respectively. Compared to patients with a CCS between 0 and 2, the highest bio-ADM result (p = 0.01) was observed in patients with a CCS of 3 and in patients with a vena cava index > 1 (p = 0.05), suggesting a potential role for bio-ADM in detecting congestion, which is considered the leading cause of death and rehospitalization for patients with heart failure. Furthermore, higher levels of bio-ADM were linked to increased furosemide treatment and a higher hospital mortality rate, likely related to the amount of congestion (p = 0.002 and p < 0.001, respectively). Petition 870230018821, dated 06 / 03 / 2023, pages 105 / 138 98 / 98 Variable All (n=168) Demographic Range Sex - male 45% Age - mean [IQR] 80 [73 - 86] Concomitant Diseases Heart failure - yes 39% Hypertension - yes 74% Diabetes - yes 36% Renal dysfunction - yes 24% Other Creatinine (mg / dL) - mean [IQR] 1.1 [0.9 - 3.3] Final diagnosis - pneumonia 17% Final diagnosis - AHF 82% In-hospital mortality 10% Table 22: Patient characteristics.

[00246] Bio-ADM was also associated with clinical congestion. The clinical congestion score combines the presence of peripheral edema, orthopnea, and jugular venous pressure > 6 cm (each adding one point to the score). BioADM was elevated in patients with a clinical congestion score (CCS) of 3 (n = 30, p < 0.01, Figure 5). Petition 870230018821, dated 06 / 03 / 2023, pages 106 / 138

Claims

1 / 8 CLAIMS 1. An in vitro method for a) diagnosing congestion or assessing or monitoring the extent of congestion in an individual or, b) predicting or determining or monitoring the need for congestion therapy or intervention or predicting or determining or monitoring the success of a congestion therapy or intervention or guiding a congestion therapy or intervention in an individual or, c) predicting decongestion or residual congestion after congestion therapy or intervention in an individual or, d) assessing decongestion or residual congestion after congestion therapy or intervention in an individual or, e) evaluating the decision regarding hospital discharge of an individual,characterized by the fact that said individual is an individual who has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF or in which said individual is an individual who has chronic heart failure with worsening signs / symptoms of chronic heart failure, and in which Pro-Adrenomedullin or fragments thereof of at least 5 amino acids is used as a marker for congestion, and in which said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRproADM according to SEQ ID NO: 3 or mature ADM-NH2 according to Petition 870230018821, dated 06 / 03 / 2023, p. 107 / 138 2 / 8 with SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CT-proADM according to SEQ ID NO: 6., 2. A method according to claim 1, characterized in that it comprises the steps of: • determining the level of Pro-Adrenomedullin or fragments thereof of at least 5 amino acids in a body fluid obtained from said individual; and a) correlating said level of Pro-Adrenomedullin or fragments thereof with the extent of congestion in said individual or diagnosing congestion, wherein a level elevated above a certain threshold is indicative of congestion or the extent of congestion, or b) correlating said level of Pro-Adrenomedullin or fragments thereof with the success of a congestion therapy or intervention in said individual, wherein a level below a certain threshold is predictive of successful congestion therapy or intervention, and wherein a level above a certain threshold is indicative of the need for congestion therapy or intervention,or c) correlate the aforementioned level of Pro-Adrenomedullin or fragments thereof with a prognosis of decongestion or residual congestion after therapy or congestion intervention, wherein a level elevated above a certain limit indicates residual congestion after therapy or congestion intervention, while a level below a certain limit indicates decongestion after therapy or congestion intervention, or d) correlate the aforementioned level of Pro-Adrenomedullin or Petition 870230018821, dated 06 / 03 / 2023, page 108 / 138 3 / 8 fragments thereof with decongestion or residual congestion after therapy or congestion intervention, wherein a level elevated above a certain limit indicates residual congestion after therapy or congestion intervention, while a level below a certain limit indicates decongestion after therapy or congestion intervention,or e) Correlate the aforementioned level of Pro-Adrenomedullin or fragments thereof with the assessment of the decision regarding hospital discharge, wherein a level elevated above a certain limit means that the individual would not be discharged and wherein a level below a certain limit means that the individual may be discharged, wherein said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRproADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CT-proADM according to SEQ ID NO:

6.

3. Method, according to claim 1, characterized in that it comprises the steps of: • determining the level of immunoreactive analyte through the use of at least one ligand that binds to a region within the amino acid sequence of Pro-Adrenomedullin or fragments thereof of at least 5 amino acids in a body fluid obtained from said individual; and a) correlating said level of immunoreactive analyte with the extent of congestion in said individual or diagnosing congestion, wherein a level elevated above a certain threshold is indicative of congestion or the extent of congestion, or b) correlating said level of immunoreactive analyte with the success of a congestion therapy or intervention in said individual, wherein a level below a certain threshold is predictive of successful congestion therapy or intervention,and where a level above a certain threshold is indicative of the need for congestion therapy or intervention, or c) correlate said immunoreactive analyte level with a prognosis of decongestion or residual congestion after congestion therapy or intervention, where a level elevated above a certain threshold indicates residual congestion after congestion therapy or intervention, while a level below a certain threshold indicates decongestion after therapy, or d) correlate said immunoreactive analyte level with decongestion or residual congestion after congestion therapy or intervention, where a level elevated above a certain threshold indicates residual congestion after congestion therapy or intervention, while a level below a certain threshold indicates decongestion after congestion therapy or intervention,or e) correlate the aforementioned immunoreactive analyte level with the assessment of the decision regarding hospital discharge, where a level elevated above a certain limit means that the individual would not be discharged and where a level below a certain limit means that the individual can be discharged, wherein said Pro-Adrenomedullin or fragment is selected from the group comprising Pro-Adrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MRproADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CT-proADM according to SEQ ID NO:

6.

4. Method, according to any one of claims 1 to 3, characterized in that the said extent of congestion is expressed as a congestion score, in particular a clinical congestion score.

5. A method, according to any one of claims 1 to 4, characterized in that the individual is stratified: a) into groups of congestion degrees, or b) into non-responder and / or responder and / or poor responder to congestion therapy or intervention, or c) into a group with decongestion or into a group with residual congestion after congestion therapy or intervention.

6. Method, according to any one of claims 1 to 5, characterized in that said fragment can be selected from MR-proADM according to SEQ ID NO: 3 or mature ADMNH2 according to SEQ ID NO:

4.

7. A method, according to any one of claims 1 to 6, characterized in that the level of Pro-Adrenomedullin or fragments thereof of at least 5 amino acids is determined by using a ligand for Pro-Adrenomedullin or fragments thereof of at least 5 amino acids.

8. Method, according to claim 6, characterized by the fact that the ligand is selected from the group comprising an antibody, an antibody fragment or a non-Ig structure that binds to Pro-Adrenomedullin or fragments thereof of at least 5 amino acids.

9. Method, according to any one of claims 1 to 8, characterized in that said limit is within a limit range that is a limit range for mature plasma ADM-NH2 according to SEQ ID NO: 4 between 50 and 100 pg / mL and for plasma MRproADM between 0.5 and 1.5 nmol / L and for plasma CT-proADM between 85 and 350 pmol / L.

10. Method, according to any one of claims 1 to 9, characterized in that said determination of ProAdrenomedullin or fragments thereof of at least 5 amino acids is performed more than once in a patient.

11. A method, according to any one of claims 1 to 10, characterized in that the sample is taken at hospital admission or at hospital discharge.

12. Method, according to any one of claims 1 to 11, characterized in that said monitoring is performed in order to evaluate the response of said individual to the preventive and / or therapeutic measures taken.

13. Method, according to any one of claims 1 to 12, characterized in that it is for stratifying said individuals into groups according to degree of congestion.

14. Method, according to any one of claims 1 to 13, characterized in that said level of ProAdrenomedullin or fragments thereof or said level of immunoreactive analyte is correlated with a risk of death or an adverse event in an individual with acute heart failure, wherein a level elevated above a certain threshold is predictive of a higher risk of death or adverse events. Petition 870230018821, dated 06 / 03 / 2023, p. 112 / 138 7 / 8 15. A method, according to any one of claims 1 to 14, characterized in that said level of Pro-Adrenomedullin or fragments thereof or said level of immunoreactive analyte is used to guide congestion therapy or intervention, wherein the therapy or intervention is indicated if said level of Pro-Adrenomedullin or fragments is above a certain threshold, and wherein congestion therapy or intervention is not indicated if said level of Pro-Adrenomedullin or fragments is below a certain threshold.

16. Method, according to any one of claims 1 to 15, characterized in that said level of ProAdrenomedullin or fragments thereof or said level of immunoreactive analyte is used to determine the need for congestion therapy or intervention.

17. A method, according to any one of claims 1 to 16, characterized in that said intervention or therapy is selected from the group comprising the administration of diuretics, administration of inotropes, administration of vasodilators, ultrafiltration.

18. Use of Pro-Adrenomedullin or fragments thereof of at least 5 amino acids, characterized by the fact that it acts as a marker for congestion, to: a) diagnose congestion or assess or monitor the extent of congestion in an individual, or b) predict or determine or monitor the need for congestion therapy or intervention or predict or determine or monitor the success of a congestion therapy or intervention or guide a congestion therapy or intervention in an individual, or Petition 870230018821, dated 06 / 03 / 2023, p.113 / 138 8 / 8 c) to predict residual decongestion or congestion after congestion therapy or intervention in an individual, or d) to assess residual decongestion or congestion after congestion therapy or intervention in an individual, or e) to assess the decision regarding hospital discharge of an individual, wherein said individual has acute heart failure that is newly onset AHF or acute decompensated HF or acute decompensated chronic HF or wherein said individual has chronic heart failure with worsening signs / symptoms of chronic heart failure, and wherein said Pro-Adrenomedullin or fragments of at least 5 amino acids is selected from the group comprising ProAdrenomedullin according to SEQ ID NO: 1 or PAMP according to SEQ ID NO: 2 or MR-proADM according to SEQ ID NO: 3 or mature ADM-NH2 according to SEQ ID NO: 4 or ADM-Gly according to SEQ ID NO: 5 or CT-proADM according to SEQ ID NO: 6.Petition 870230018821, dated 06 / 03 / 2023, pp. 114 / 138.

Images