WO2019234099A1

WO2019234099A1 - Methods for diagnosing, predicting the outcome and treating a patient suffering from heart failure with preserved ejection fraction

Info

Publication number: WO2019234099A1
Application number: PCT/EP2019/064649
Authority: WO
Inventors: Patrick ROSSIGNOL; Faiez Zannad; Natalia LOPEZ-ANDRES; Walter J. PAULUS
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université De Lorraine; Centre Hospitalier Et Universitaire De Nancy (Chu); Navarrabiomed Fundación Miguel Servet
Priority date: 2018-06-06
Filing date: 2019-06-05
Publication date: 2019-12-12

Abstract

The invention relates to methods for diagnosing and predicting the outcome of patient suffering from heart failure with preserved ejection fraction (HFpEF) and methods for the treatment of HFpEF. HFpEF still lacks evidence-based therapies. There is currently no single biomarker that can accurately predict the development of left ventricular diastolic dysfunction (DD) and HFpEF outcomes. In four prospective multicenter patient cohorts with a metabolic risk profile (METR-DHF cohort, n=1428), the inventors selected biomarkers associated with echocardiography patterns of DD. The association of these selected biomarkers with the outcome of cardiovascular hospitalizations or cardiovascular death at 12 months was further investigated in an independent prospective cohort of patients with HFpEF (MEDIA-DHF cohort, n=533). The inventors also investigated in human fibroblasts the mechanistic significance of the selected biomarker. In multivariate analysis, Intercellular Adhesion Molecule 3 (ICAM-3) and NT-proBNP were found associated with DD in METR-DHF. Plasma NT-proBNP > 300 pg/ml (HR 13.5 [1.82 - 100], p=0.011) and ICAM-3 > 150 ng/ml (HR 3.01 [1.14 - 7.95], p=0.026) were found associated with the primary outcome (30/ 437) the prospective cohort.. In human cardiac fibroblasts ICAM-3 treatment induced a fibrosis phenotype and mediated the pro-fibrotic effects of angiotensin II and aldosterone. Besides the expected NT-proBNP, ICAM-3 blood concentration is associated with DD in patients at risk of HF and is predictive of CV outcomes in patients with HFpEF. Thus, the invention relates to a method of diagnosing and predicting the outcome of a patient suffering from HFpEF comprising determining the level of ICAM-3 and the level of NT-proBNP in a blood sample (e.g. by ELISA) obtained from the patient and ICAM-3 inhibitor for use in the treatment of HFpEF.

Description

METHODS FOR DIAGNOSING, PREDICTING THE OUTCOME AND TREATING A PATIENT SUFFERING FROM HEART FAILURE WITH PRESERVED

EJECTION FRACTION

FIELD OF THE INVENTION:

The present invention relates to methods for predicting the outcome and the treatment of a patient suffering from heart failure with preserved ejection fraction.

BACKGROUND OF THE INVENTION:

Heart failure with preserved ejection fraction (HFpEF), formerly called Diastolic Heart Failure (DHF), accounts for more than 50% of all heart failure cases and portrays a high risk of morbidity and mortality (1). A better prevention of HFpEF and its complications is an essential public health goal, although both the prediction and diagnosis of this disorder still remain uncertain. The diagnosis of HFpEF is based on signs or symptoms of heart failure, normal or mildly abnormal left ventricular ejection fraction (FVEF) and evidence of diastolic left ventricular (FV) dysfunction (2). While natriuretic peptides (BNP or NT-proBNP) are correlated with symptomatic FV diastolic dysfunction, the concentration of these biomarkers can vary with age, sex, bodyweight as well as several comorbidities (3-6). Thus, elevated BNP or pro-BNP values do not represent standalone evidence for symptomatic FV diastolic dysfunction and thus additional non-invasive echocardiography tests are required to confirm the diagnosis (2). Contrary to their usefulness for the diagnosis of symptomatic diastolic FV dysfunction, natriuretic peptides represent sub-optimal screening tests for pre-clinical diagnosis of FV dysfunction (7). As a result, there is currently no single biomarker that can accurately predict the development of FV diastolic dysfunction and HFpEF outcomes. This is likely due to the fact that pathophysiology of HFpEF is complex and still incompletely understood. The inventors have designed a two-step clinical research program with the objective of identifying biomarkers associated with diastolic dysfunction in asymptomatic patients at risk of HFPEF and predictive of outcome in patients with symptomatic HFPEF. The inventors selected biomarkers ontologically associated with the processes of neurohormonal activation, extracellular matrix turnover, cytokine activation, cardiomyocyte stress, cell adhesion and oxidative stress, all reported as potentially relevant in HFpEF pathophysiology (8,9). In a separate set of in vitro experiments, the inventors have also investigated the role of one of the selected biomarkers as a biotarget. SUMMARY OF THE INVENTION:

The present invention relates to methods and kits for diagnosing and predicting the outcome of a patient suffering from heart failure with preserved ejection fraction (HFpEF).

The present invention also relates to methods and pharmaceutical compositions for the treatment of HFpEF.

In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Heart failure with preserved ejection fraction (HFpEF) still lacks evidence-based therapies. There is currently no single biomarker that can accurately predict the development of left ventricular diastolic dysfunction and HFpEF outcomes. In four prospective multicenter patient cohorts with a metabolic risk profile (METR-DHF cohort, n=l428), the inventors selected biomarkers associated with echocardiography patterns of diastolic dysfunction, among 28 candidate pathophysiologically relevant biomarkers. The association of these selected biomarkers with the outcome of cardiovascular hospitalizations or cardiovascular death at 12 months was further investigated in an independent prospective cohort of patients with HFpEF (MEDIA-DHF cohort, n=533). Finally the inventors investigated in human fibroblasts the mechanistic significance of one molecule found to be diagnostically associated with diastolic dysfunction and prognostically predictive of CV outcome. In multivariate analysis, four biomarkers were found associated with diastolic dysfunction in METR-DHF: PICP per 50 ng/ml increment (HR 1.50 (0.44 - 0.63) r=0.016), NT-proBNP pg/ml > 57 pg/ml (HR 2.46 (1.27 - 2.63), p=0.008), E-selectin per 10 ng/ml (HR 0.72 (0.17 - 0.22), r=0.010) and Intercellular Adhesion Molecule 3 (ICAM-3) > 0.32 ng/ml (HR 3.08 (1.35 - 2.41), pO.OOOl). Plasma NT-proBNP > 300 pg/ml (HR 13.5 [1.82 - 100], p=0.011) and ICAM-3 > 150 ng/ml (HR 3.01 [1.14 - 7.95], p=0.026) were furthermore found associated with the primary outcome (30/437) in the prospective cohort. In human cardiac fibroblasts, treatment with recombinant ICAM-3 increased profibrotic markers and mediated the pro-fibrotic effects of angiotensin II and aldosterone. Besides the expected NT-proBNP, ICAM-3 blood concentration is associated with diastolic dysfunction in patients at risk of HF and is predictive of CV outcomes in patients with HFpEF. In vitro experiments suggest that ICAM-3 may represent a relevant therapeutic target. These results serve as the basis for a potential precision medicine approach targeting patients with an ICAM-3 bioprofile with an anti-ICAM-3 agent.

A first object of the invention relates to a method of identifying a patient having or at risk of having or developing heart failure with preserved ejection fraction comprising a step of determining the level of ICAM-3 and the level of NT-proBNP in a blood sample obtained from the patient.

The method of the invention may further comprise a step consisting of comparing the level of ICAM-3 in the blood sample with a reference value 1, wherein detecting differential in the level of ICAM-3 between the blood sample and the reference value 1 is indicative of patient having or at risk of having or developing HFpEF.

The method of the invention may further comprise a step consisting of comparing the level of NT-proBNP in the blood sample with a reference value 2, wherein detecting differential in the level of NT-proBNP between the blood sample and the reference value 2 is indicative of patient having or at risk of having or developing HFpEF.

In a particular embodiment, the level of ICAM-3 and the level of NT-proBNP are determined simultaneously.

In some embodiments, the present invention relates to a method of identifying a patient having or at risk of having or developing heart failure with preserved ejection fraction comprising the steps of: i) determining the level of ICAM-3 and the level of NT-proBNP in a blood sample obtained from the patient, ii) comparing the level determined at step i) with a reference value 1 for ICAM-3 and a reference value 2 for a NT-proBNP, wherein detecting differential in the level of ICAM-3 and in the level of NT-proBNP between the blood sample and the reference value 1 and the reference 2 is indicative of patient having or at risk of having or developing HFpEF.

As used herein, the term "heart failure with preserved ejection fraction” has its general meaning in the art and refers to a complex syndrome characterized by heart failure (HF) signs and symptoms and a normal or near-normal left ventricular ejection fraction (FVEF). More specific diagnostic criteria include signs/symptoms of HF, objective evidence of diastolic dysfunction, disturbed left ventricular (FV) filling, structural heart disease, and elevated brain natriuretic peptides. Additional cardiac abnormalities can include subtle alterations of systolic function, impaired atrial function, chronotropic incompetence, or haemodynamic alterations, such as elevated pre-load volumes.

The term“a patient at risk of having or developing heart failure with preserved ejection fraction” refers to a patient afflicted with diastolic dysfunction or left ventricular diastolic dysfunction without clinical signs of heart failure. The term“a patient at risk of having or developing heart failure with preserved ejection fraction” also refers to asymptomatic patients without clinical signs of heart failure. As used herein the term“ICAM-3” has its general meaning in the art and refers to the intercellular adhesion molecule 3 encoded by the ICAM3 gene (Gene ID: 3385). An exemplary amino acid sequence is the NCBI Reference Sequence: NP 001307534.1.

As used herein the term“NT-proBNP” also known as N-terminal prohormone of brain natriuretic peptide has its general meaning in the art and refers to a prohormone with a 76 amino acid N-terminal inactive protein that is cleaved from the molecule to release brain natriuretic peptide.

As used herein, the term“blood sample” refers to a whole blood sample, serum sample and plasma sample. A blood sample may be obtained by methods known in the art including venipuncture or a finger stick. Serum and plasma samples may be obtained by centrifugation methods known in the art. The sample may be diluted with a suitable buffer before conducting the assay.

A predetermined reference value can be relative to a number or value derived from population studies, including without limitation, patient having or at risk of having or developing heart failure with preserved ejection fraction, a patient not having or not at risk of having or developing heart failure with preserved ejection fraction, subjects having similar body mass index, total cholesterol levels, LDL/HDL levels, systolic or diastolic blood pressure, subjects of the same or similar age range, subjects in the same or similar ethnic group, and subjects having the same severity of heart failure. Such predetermined reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of metabolic syndrome. In some embodiments, the predetermined reference values 1 are derived from the level of ICAM-3 in a control sample derived from one or more subjects who were not subjected to the event. Furthermore, retrospective measurement of the level of ICAM-3 in properly banked historical subject samples may be used in establishing these predetermined reference values.

In some embodiments, the predetermined reference values 2 are derived from the level of NT-proBNP in a control sample derived from one or more subjects who were not subjected to the event. Furthermore, retrospective measurement of the level of NT-proBNP in properly banked historical subject samples may be used in establishing these predetermined reference values.

The predetermined reference value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the predetermined reference value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of the marker in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured levels of the marker in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. S AS, C RE ATE -ROC. S AS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In one embodiment, the reference value 1 may correspond to the expression level of IC AM-3 determined in a blood sample associated with a patient having or at risk of having or developing heart failure with preserved ejection fraction. Accordingly, a higher or equal expression level of IC AM-3 than the reference value is indicative of a patient having or at risk of having or developing heart failure with preserved ejection fraction, and a lower expression level of ICAM-3 than the reference value is indicative of a patient not having or not at risk of having or developing heart failure with preserved ejection fraction.

In another embodiment, the reference value 1 may correspond to the expression level of ICAM-3 determined in a blood sample associated with a patient not having or not at risk of having or developing heart failure with preserved ejection fraction. Accordingly, a higher expression level of ICAM-3 than the reference value 1 is indicative of a patient having or at risk of having or developing heart failure with preserved ejection fraction, and a lower or equal expression level of ICAM-3 than the reference value 1 is indicative of a patient not having or not at risk of having or developing heart failure with preserved ejection fraction.

In one embodiment, the reference value 2 may correspond to the expression level of NT-proBNP determined in a blood sample associated with a patient having or at risk of having or developing heart failure with preserved ejection fraction. Accordingly, a higher or equal expression level of NT-proBNP than the reference value 2 is indicative of a patient having or at risk of having or developing heart failure with preserved ejection fraction, and a lower expression level of NT-proBNP than the reference value 2 is indicative of a patient not having or not at risk of having or developing heart failure with preserved ejection fraction.

In another embodiment, the reference value 2 may correspond to the expression level of NT-proBNP determined in a blood sample associated with a patient not having or not at risk of having or developing heart failure with preserved ejection fraction. Accordingly, a higher expression level of NT-proBNP than the reference value 2 is indicative of a patient having or at risk of having or developing heart failure with preserved ejection fraction, and a lower or equal expression level of NT-proBNP than the reference value 2 is indicative of a patient not having or not at risk of having or developing heart failure with preserved ejection fraction.

In some embodiments, the invention relates to a method of identifying a patient having or at risk of having or developing heart failure with preserved ejection fraction, comprising the steps of: i) determining the expression level of ICAM-3 and of NT-proBNP in a blood sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value 1 for ICAM-3 and a predetermined reference value 2 for NT- proBNP, and iii) concluding that the patient is having or at risk of having or developing heart failure with preserved ejection fraction when the levels determined at step i) are higher than the predetermined reference value 1 and the predetermined reference value 2, or concluding that the patient is not having or not at risk of having or developing heart failure with preserved ejection fraction when the levels determined at step i) are lower than the predetermined reference value 1 and predetermined reference value 2.

In a further aspect, the present invention relates to a method of predicting the outcome of a patient suffering from heart failure with preserved ejection fraction comprising the steps of: i) determining the level of ICAM-3 and the level of NT-proBNP in a blood sample obtained from the patient, ii) comparing the level determined at step i) with a predetermined reference value 1 for ICAM-3 and a predetermined reference value 2 for NT-proBNP and iii) detecting differential between the levels determined at step i) with the predetermined reference value 1 and the predetermined reference value 2 indicates the outcome of the patient.

The method of the present invention is particularly suitable for predicting an event consisting of cardiovascular hospitalization of the patient. As used herein, the term “cardiovascular hospitalization” includes hospitalization due to heat failure and hospitalizations due to other cardiovascular causes. The method of the present invention is also particularly suitable for predicting the outcome which is cardiovascular death of the patient. As used herein, the term“cardiovascular death” includes sudden cardiac death, death due to heart failure, and death due to other cardiovascular causes.

Typically, the levels of ICAM-3 and of NT-proBNP are deemed to be higher than the predetermined reference 1 for ICAM-3 and the predetermined reference 2 for NT-proBNP when it is concluded that the patient has a high risk of being hospitalized or of dying. Accordingly, in some embodiments, it is concluded that the patient is at risk of dying or being hospitalized when the levels determined at step i) are higher than the predetermined reference value 1 and the predetermined reference value 2.

In one embodiment, the reference value 1 may correspond to the expression level of ICAM-3 and the reference value 2 may correspond to the expression level of NT- proBNP determined in a blood sample associated with a patient suffering from heart failure with preserved ejection fraction having high risk of being hospitalized or of dying. Accordingly, a higher or equal expression level of ICAM-3 than the reference value 1 and a higher or equal expression level of NT-proBNP than the reference value are indicative of a patient having high risk of being hospitalized or of dying, and a lower expression level of ICAM-3 than the reference value 1 and a lower expression level of NT-proBNP than the reference value 2 are indicative of a patient not at risk of being hospitalized or of dying.

In another embodiment, the reference value 1 may correspond to the expression level of ICAM-3 and the reference value 2 may correspond to the expression level of NT-proBNP determined in a blood sample associated with a patient suffering from heart failure with preserved ejection fraction not at risk of being hospitalized or of dying. Accordingly, a higher expression level of ICAM-3 than the reference value 1 and a higher expression level of NT- proBNP than the reference value 2 are indicative of a patient having high risk of being hospitalized or of dying, and a lower or equal expression level of ICAM-3 than the reference value 1 and a lower or equal expression level of NT-proBNP than the reference value 2 are indicative of a patient not at risk of being hospitalized or of dying. In some embodiments, the invention relates to a method of identifying a patient suffering from heart failure with preserved ejection fraction not at risk of being hospitalized or of dying, comprising the steps of: i) determining the expression level of ICAM-3 and of NT- proBNP in a blood sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value 1 for ICAM-3 and a predetermined reference value 2 for NT-proBNP, and iii) concluding that the patient is having high risk of being hospitalized or of dying when the levels determined at step i) are higher than the predetermined reference value 1 and the predetermined reference value 2, or concluding that the patient is not at risk of being hospitalized or of dying when the level determined at step i) is lower than the predetermined reference value 1 and the predetermined reference value 2 .

When it is determined that the patient is having or at risk of having or developing heart failure with preserved ejection fraction or that the patient is at risk of dying or being hospitalized, the patient is subsequently administered with a therapeutically effective amount of a drug suitable for the treatment and prevention of heart failure. Typically said drug is selected from the group consisting of angiotensin-receptor blocker (ARB), ARB-neprilysin inhibitor, mineralocorticoid receptor antagonist (e.g. spironolactone), SGLT-2 inhibitors, diuretics, angiotensin converting enzyme (ACE) inhibitors, digoxin (also called digitalis), calcium channel blockers, and beta-blockers. In mild cases, thiazide diuretics, such as hydrochlorothiazide at 25-50 mg/day or chlorothiazide at 250-500 mg/day, are useful. In particular diuretics (Loop diuretics or thiazides diuretics) are useful in congested and /or hypertensive patients. However, supplemental potassium chloride may be needed, since chronic diuresis causes hypokalemis alkalosis. Typical doses of ACE inhibitors include captopril at 25- 50 mg/day and quinapril at 10 mg/day. In some embodiments, the subject is administered with an adrenergic beta-2 agonist. An "adrenergic beta-2 agonist" refers to adrenergic beta-2 agonists and analogues and derivatives thereof, including, for example, natural or synthetic functional variants which have adrenergic beta-2 agonist biological activity, as well as fragments of an adrenergic beta-2 agonist having adrenergic beta-2 agonist biological activity. Commonly known adrenergic beta-2 agonists include, but are not limited to, clenbuterol, albuterol, formeoterol, levalbuterol, metaproterenol, pirbuterol, salmeterol, and terbutaline. In some embodiments, the subject is administered with an adrenergic beta-l antagonist. Adrenergic beta-l antagonists and adrenergic beta-l blockers refer to adrenergic beta-l antagonists and analogues and derivatives thereof, including, for example, natural or synthetic functional variants which have adrenergic beta-l antagonist biological activity, as well as fragments of an adrenergic beta-l antagonist having adrenergic beta-l antagonist biological activity. Commonly known adrenergic beta-l antagonists include, but are not limited to, acebutolol, atenolol, betaxolol, bisoprolol, esmolol, and metoprolol.

The measurement of the level of ICAM-3 or NT-proBNP in the blood sample is typically carried out using standard protocols known in the art. For example, the method may comprise contacting the blood sample with a binding partner capable of selectively interacting with ICAM-3 or with NT-proBNP in the sample. In some embodiments, the binding partners are antibodies, such as, for example, monoclonal antibodies or even aptamers. For example the binding may be detected through use of a competitive immunoassay, a non-competitive assay system using techniques such as western blots, a radioimmunoassay, an ELISA (enzyme linked immunosorbent assay), a“sandwich” immunoassay, an immunoprecipitation assay, a precipitin reaction, a gel diffusion precipitin reaction, an immunodiffusion assay, an agglutination assay, a complement fixation assay, an immunoradiometric assay, a fluorescent immunoassay, a protein A immunoassay, an immunoprecipitation assay, an immunohistochemical assay, a competition or sandwich ELISA, a radioimmunoassay, a Western blot assay, an immunohistological assay, an immunocytochemical assay, a dot blot assay, a fluorescence polarization assay, a scintillation proximity assay, a homogeneous time resolved fluorescence assay, a IAsys analysis, and a BIAcore analysis. The aforementioned assays generally involve the binding of the partner (ie. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. An exemplary biochemical test for identifying specific proteins employs a standardized test format, such as ELISA test, although the information provided herein may apply to the development of other biochemical or diagnostic tests and is not limited to the development of an ELISA test (see, e.g., Molecular Immunology: A Textbook, edited by Atassi et al. Marcel Dekker Inc., New York and Basel 1984, for a description of ELISA tests). Therefore ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize ICAM-3 or NT-proBNP. A sample containing or suspected of containing ICAM-3 or NT- proBNP is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art. Measuring the level of IC AM-3 or NT-proBNP (with or without immunoassay-based methods) may also include separation of the compounds: centrifugation based on the compound’s molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the compound's affinity for the particular solid-phase that is used. Once separated, said one or two biomarkers proteins may be identified based on the known "separation profile" e.g., retention time, for that compound and measured using standard techniques. Alternatively, the separated compounds may be detected and measured by, for example, a mass spectrometer. Typically, levels of immunoreactive ICAM-3 or NT-proBNP in a sample may be measured by an immunometric assay on the basis of a double-antibody "sandwich" technique, with a monoclonal antibody specific for ICAM-3 or NT-proBNP (Cayman Chemical Company, Ann Arbor, Michigan). According to said embodiment, said means for measuring ICAM-3 level and NT-proBNP level are for example i) a ICAM-3 buffer and a NT-proBNP buffer, ii) a monoclonal antibody that interacts specifically with ICAM-3 and a monoclonal antibody that interacts specifically with NT-proBNP, iii) an enzyme-conjugated antibody specific for ICAM-3 and a predetermined reference value 1 of ICAM-3 and an enzyme-conjugated antibody specific for NT-proBNP and a predetermined reference value 2 of NT-proBNP.

A further object of the invention relates to an ICAM-3 inhibitor for use in the treatment of HFpEF in a patient in need thereof.

In a further aspect, the present invention relates to an ICAM-3 inhibitor for use in the treatment of HFpEF in a patient in need thereof, wherein the patient was being classified as at risk of dying or being hospitalized by the method as above described.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patients at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

The term“ICAM-3 inhibitor” has its general meaning in the art and refers to a compound that selectively blocks or inactivates the ICAM-3. The term“ICAM-3 inhibitor” also refers to a compound that selectively blocks the ICAM-3 adhesion property and downstream effectors such as inhibiting the collagen type I secretion, fibronectin expression, MMP-l secretion and IL-6 secretion. The term“ICAM-3 inhibitor” also refers to a compound able to prevent the action of ICAM-3 for example by inhibiting the extracellular matrix remodeling, inflammation, profibrotic stimuli and monocyte recruitment. As used herein, the term “selectively blocks or inactivates” refers to a compound that preferentially binds to and blocks or inactivates ICAM-3 with a greater affinity and potency, respectively, than its interaction with the other sub-types of the ICAM family. Compounds that block or inactivate ICAM-3, but that may also block or inactivate other ICAM sub-types, as partial or full inhibitors, are contemplated. The term“ICAM-3 inhibitor” also refers to a compound that inhibits ICAM-3 expression. Typically, an ICAM-3 inhibitor is a small organic molecule, a polypeptide, an aptamer, an antibody, an oligonucleotide or a ribozyme.

Tests and assays for determining whether a compound is an ICAM-3 inhibitor are well known by the skilled person in the art such as described in Humeres et ah, 2016 (21) and in the example.

In one embodiment of the invention, ICAM-3 inhibitors include but are not limited to the anti-ICAM-3 antibodies such as monoclonal antibodies CAL 3.10; CAL 3.38; CAL 3.41; BY 44; 186-269; B-P12; AZN-ICAM-3.1; BU68; 3A9 (Gregory et a , 1998; Moffatt et a , 1999) , shRNA, siRNA and compounds described in Gregory et al., 1998; Moffatt et al., 1999; WO2012/046001; and US5891841.

In another embodiment, the ICAM-3 inhibitor of the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then after raising aptamers directed against ICAM- 3 of the invention as above described, the skilled man in the art can easily select those blocking or inactivating ICAM-3.

In another embodiment, the ICAM-3 inhibitor of the invention is an antibody (the term including“antibody portion”) directed against ICAM-3.

In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of IC AM-3. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

Briefly, the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in ICAM-3. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modem Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab’)2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc’ regions to produce a functional antibody.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et ah, /. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non human sequences. The present invention also includes so-called single chain antibodies.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. In a preferred embodiment, the ICAM-3 inhibitor of the invention is a Human IgG4.

In another embodiment, the antibody according to the invention is a single domain antibody. The term“single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called“nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term“VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term“complementarity determining region” or“CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation. VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen- specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the“Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The“Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In one embodiment, the ICAM-3 inhibitor of the invention is an ICAM-3 expression inhibitor.

The term“expression” when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs, which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., ICAM-3) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.

An“inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. ICAM-3 expression inhibitors for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of ICAM-3 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of ICAM-3 proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding ICAM-3 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) and short hairpin RNAs (shRNAs) can also function as ICAM-3 expression inhibitors for use in the present invention. ICAM-3 gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that ICAM-3 expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as ICAM-3 expression inhibitors for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of ICAM-3 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful a ICAM-3 inhibitors can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3’ ends of the molecule, or the use of phosphorothioate or 2’-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing ICAM-3. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman C.O., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild- type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et ah, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUCl8, pUCl9, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

Typically the inhibitors according to the invention as described above are administered to the patient in a therapeutically effective amount.

By a "therapeutically effective amount" of the inhibitor of the present invention as above described is meant a sufficient amount of the inhibitor for treating HFpEF at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the inhibitors and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific inhibitor employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific inhibitor employed; the duration of the treatment; drugs used in combination or coincidential with the specific inhibitor employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the inhibitor at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the inhibitor of the present invention for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the inhibitor of the present invention, preferably from 1 mg to about 100 mg of the inhibitor of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

In some embodiments, the ICAM-3 inhibitor of the present invention is administered to the patient in combination with a drug suitable for the treatment and prevention of heart failure.

In some embodiments, the ICAM-3 inhibitor of the invention is administered sequentially or concomitantly with a drug suitable for the treatment and prevention of heart failure. The inhibitors of the invention may be used or prepared in a pharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical composition comprising the inhibitor of the invention and a pharmaceutical acceptable carrier for use in the treatment of HFpEF in a patient of need thereof.

Typically, the inhibitor of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, intramuscular, intravenous, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, intraperitoneal, intramuscular, intravenous and intranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze- dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising inhibitors of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The inhibitor of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active inhibitors in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the patient being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual patient.

In addition to the inhibitors of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

Pharmaceutical compositions of the invention may include any further compound which is used in the treatment of heart failure.

In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of heart failure with preserved ejection fraction in a patient in need thereof, wherein the patient was being classified as at risk of dying or being hospitalized by the method as above described.

The invention also provides kits comprising the inhibitor of the invention. Kits containing the inhibitor of the invention find use in therapeutic methods.

A further aspect of the invention relates to a method of treating heart failure with preserved ejection fraction in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the ICAM-3 inhibitor of the invention.

A further aspect of the invention relates to a method for treating heart failure with preserved ejection fraction in a patient in need thereof comprising the steps of:

a) determining whether the patient is having or at risk of having or developing heart failure with preserved ejection fraction by performing the method according to the invention, and

b) administering the ICAM-3 inhibitor if said patient has been considered as having or at risk of having or developing heart failure with preserved ejection fraction.

A further aspect of the invention relates to a method for treating heart failure with preserved ejection fraction in a patient in need thereof comprising the steps of: a) determining whether the patient is at risk of dying or being hospitalized by performing the method according to the invention, and

b) administering the ICAM-3 inhibitor if said patient has been considered as at risk of dying or being hospitalized.

In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for treating heart failure with preserved ejection fraction in a patient in need thereof, wherein the method comprises the steps of:

providing an ICAM3, ICAM-3 expressing cell, providing a cell, tissue sample or organism expressing ICAM-3, cardiac fibroblast, vascular smooth muscle cell and macrophage,

providing a candidate compound such as a small organic molecule, a polypeptide, an aptamer, an antibody or an intra-antibody,

measuring the ICAM-3 activity,

and selecting positively candidate compounds that inhibit ICAM-3 activity.

Methods for measuring ICAM-3 activity are well known in the art (Humeres et ah, 2016). For example, measuring the ICAM-3 activity involves determining the Ki of ICAM-3 cloned and transfected in a stable manner into a CHO cell line, cardiac fibroblast, vascular smooth muscle cell and macrophage, measuring collagen type I, fibronectin, MMP-l, TIMP-l and IL-6, measuring pro-fibrotic stimuli, measuring monocyte recruitment, and assessing inflammation and extracellular matrix remodeling in the present or absence of the candidate compound.

Tests and assays for screening and determining whether a candidate compound is a ICAM-3 inhibitor are well known in the art (Humeres et ah, 2016). In vitro and in vivo assays may be used to assess the potency and selectivity of the candidate compounds to inhibit ICAM- 3 activity.

Activities of the candidate compounds, their ability to bind ICAM-3 and their ability to inhibit ICAM-3 activity may be tested using isolated cardiac fibroblast, vascular smooth muscle cell and macrophage or CHO cell line cloned and transfected in a stable manner with the human ICAM-3.

Activities of the candidate compounds and their ability to bind to the ICAM-3 may be assessed by the determination of the Ki of ICAM-3 cloned and transfected in a stable manner into a CHO cell line, cardiac fibroblast, vascular smooth muscle cell and macrophage, measuring pro-fibrotic stimuli, measuring monocyte recruitment, and assessing inflammation and extracellular matrix remodeling in the present or absence of the candidate compound. The ability of the candidate compounds to inhibit ICAM-3 activity may be assessed as described in the example.

Cells expressing another ICAM than ICAM-3 may be used to assess selectivity of the candidate compounds.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Clinical (A, left panel) and Clinical and Biomarkers (B, right panel) factors associated with diastolic dysfunction in the METR-DHF cohort.

Figure 2: Survival rate according to biomarker tertiles in the MEDIA-DHF cohort (A and B).

Kaplan-Meier curves. Log-rank test adjusted on inclusion CHF status (ambulatory/recent discharge for ADHF) for biomarkers

Figure 3: mechanistic studies in human fibroblasts.

(A) Representative photographs of human myocardial sections double-stained with ICAM-3 and a-SMA (left panel) and with ICAM-3 and cd68 (right panel). (B) Extracellular matrix proteins and IL-6 secretion in human cardiac fibroblasts treated with recombinant ICAM-3 (lO ⁷M). (C) Effects of ICAM-3 silencing on ICAM-3, extracellular matrix proteins and IL-6 secretion in human cardiac fibroblasts treated with Ang II (lO ⁷M). (D) Effects of ICAM-3 silencing on ICAM-3, extracellular matrix proteins and IL-6 secretion in human cardiac fibroblasts treated with Aldo (lO ⁸M). All conditions were performed at least by triplicate. Histogram bars represent the mean±SEM of 4 assays, in arbitrary units normalized to Stain free gel or b-actin respectively for supernatant or protein lysate. *p<0.05 vs Control. Col-l, collagen type I; FN, fibronectin; MMP-l, metalloproteinase- 1; TIMP-l, tissue inhibitor of metalloproteinase- 1; IL-6, interleukin-6; Ang II, angiotensin II; Aldo, aldosterone.

Figure 4: METR- DHF (A) and MEDIA-DHF (B) cohorts flowcharts

Figure 5: ICAM-3 expression in human cultured cardiac cells.

ICAM-3 mRNA expression was quantified in human cardiac myocytes, human cardiac fibroblasts, valvular interstitial cells and valvular endothelial cells. Histogram bars represent the mean±SEM of 5 assays, in arbitrary units normalized to 18s, GADPH and b-actin. CM, cardiomyocytes; HCFs, human cardiac fibroblasts; VICs, valvular interstitial cells; VECs, valvular endothelial cells.

EXAMPLES: EXAMPLE 1:

Material & Methods

Cohorts

The Metabolic Risk-Diastolic Heart Failure (METR-DHF) study consisted of a retrospective cross-sectional cohort of 851 patients with 1) normal LV systolic function, defined as EF > 50% and LV end-diastolic volume index (LVEDVI) <97 ml/m2 (2), 2) echo data allowing to evaluate the presence or absence of diastolic dysfunction as defined by the 2007 European consensus statement on the diagnosis of HFNEF and 3) anthropometric and biological date allowing to evaluate the presence or absence of metabolic syndrome as defined by the NCEPIII criteria) (10). The METR-DHF cohort resulted from the merger of four European prospective cohorts of participants with various degree of metabolic syndrome and LV dysfunction prevalence (Stanislas (11,12), Pamplona (13,14), STOP-HF cohorts (15) and R2C2 cohort (16)), which included 1428 patients. Among these 1428, 575 had LVEF<50% and/or LVEDVI>97 mFm2 and 2 had missing echo data, resulting into a final sample of 851 patients.

The EU FP7 MEDIA consortium selected a set of 28 biomarkers which have been shown to have a pathophysiological link with diastolic dysfunction, with heart failure in general or with metabolic risk (Table 1). All biomarkers were measured in a central laboratory, blinded to the patient's clinical data.

The MEDIA DHF cohort was a multicenter, multinational (10 countries), observational cohort. Recruitment of patients was performed in 14 participating centers in Europe. The initial aim was to recruit a total of 750 patients, with a recruitment period of 18 months, and in which patients would be followed for one year. Inclusion criteria were as follows: patients 18 years of age or older, signs or symptoms of HF, able and willing to provide freely-given written informed consent. Exclusion criteria were as follows: patients with acute myocardial infarction, recent trauma or surgery (< 3 months), hemodynamically significant valvular disease, serious cerebrovascular disease or stroke in the last 3 months, chronic dialysis, chronic liver disease, chronic infectious (bacterial or viral) disease, any malignant concomitant diseases or a malignant disease in the last 5 years, systemic inflammatory diseases such as autoimmune diseases, connective tissue diseases or collagenosis, and pregnant women.

After standardized echocardiography and/or local BNP and NT-proBNP measurements, eligible patients with a diagnosis of diastolic dysfunction as established by the 2007 ESC guidelines (2) were included in the study replication MEDIA-DHF cohort. Two clinical modes of presentations were considered: i) patients discharged from hospital after admission for an acute HF episode or immediately after discharge (<60 days), and ii) ambulatory chronic disease patients.

Clinical outcomes were reported by telephone and recorded in the electronic case report form (including date and place). For hospitalizations, the letter of discharge was to be provided, while the death certificate was to be provided in instances of mortality. Primary outcome events (cardiovascular hospitalizations or cardiovascular death at 12 months) were adjudicated by an adjudicating committee blinded to the biomarker data.

The study protocol complied with the Declaration of Helsinki and was approved by the respective Ethics Committees of the participating institutions. All patients provided written informed consent. ClinicalTrials.gov Identifier: NCT02446327

Statistics

All analyses were performed using SAS R9.4 (SAS Institute, Cary, NC, USA). The 2- sided significance level was set at p<0.05. Time-to-event analyses were carried out using Cox regression (MEDIA-DHF). All baseline characteristics listed in table 1 were tested in the models, the final step retaining only significant factors using interactive backward selection. Pairwise comparisons were performed using Chi-Square, Fisher's exact or Mann- Whitney tests where appropriate. The added value of significant biomarkers was assessed as the integrated discriminant index (IDI) (17).

Mechanistic studies

Adult human cardiac fibroblasts (Promocell) were treated with recombinant ICAM-3. IC AM-3 -silenced cells were generated and treated with Angiotensin II or Aldosterone. Pro- fibrotic markers were measured by ELISA and Western blot.

Echocardiography

All patients underwent echocardiography and images were stored in a digital cine-loop format for off-line analysis according to the recommendations of the American and European Societies of Echocardiography relative to the cardiac chamber and right heart measurements (1)·

LV structure and function were evaluated from standard 2D views, including apical four-chamber, apical two-chamber and parasternal long- and short-axis views. Ventricular dimensions, wall thickness, mass and geometry were determined from 2D parasternal short- and long-axis views. LV volumes, stroke volume and ejection fraction were calculated using the biplane method of disks summation (modified Simpson’s rule). All cardiac chamber volumes and mass measurements were indexed to body surface area. Left atrial volume was assessed by the biplane area-length method from apical 2- and 4-chamber views at end-systole and was indexed to body surface area (LA volume index, LAVi). Left atrial area (cm²) was estimated from the apical views. If LA volume was not available, LA area was used to evaluate remodeling. Patients with either LAVi>40ml/m² or LA area>20cm² were considered to have dilated LA.

Peak velocity of early (E) and late (A) wave of transmitral flow and E-wave deceleration time (DT) were measured from the pulsed-wave Doppler obtained at the tip of mitral leaflets. The average of septal and lateral annular velocities (e’) was obtained by tissue Doppler imaging (TDI). The E/e’ ratio was calculated using the peak E-wave velocity and the average of septal and lateral. LV outflow tract (LVOT) time velocity integral, isovolumic relaxation time (IVRT) and color M-mode of early diastolic mitral inflow into the left ventricle (Flow propagation velocity, Vp) were acquired from apical four- and five-chamber views. Pulmonary venous flow (PVF) was sampled using pulsed-wave Doppler at 1 cm into the orifice of the right upper pulmonary vein.

Right ventricular function was estimated by measuring TAPSE (mm) and pulmonary arterial systolic pressure (PASP) was calculated using the peak velocity of tricuspid regurgitation (TR) and the maximum I VC diameter (I VC baseline) and respiratory variation (Ratio I VC inspiration / 1 VC baseline) measured 3 cm before merger with the right atrium.

Overall, echocardiographic data were complete in >75% of patients, except for the following variables: medial s’, Ard-Ad, S/D, IVRT, E/Vp (which were available in 59%, 63%, 50%, 65% and 51% of the population, respectively).

Results

Clinical features. The respective flowcharts of both cohorts are presented in the figure 4 along with their baseline features in table 1. Compared to the replication MEDIA-DHF participants, derivation METR-DHF participants were younger, more likely men, with lower body mass index, blood pressure, dyslipidemia, diabetes, and higher estimated glomerular filtration rate. Regarding medications, the latter were overall less frequently prescribed in METR-DHF participants compared to MEDIA-DHF participants (Table 1). Most (85%) MEDIA-DHF participants were recruited in the chronic setting (391/462) (data not shown). The primary event of cardiovascular death or heart failure hospitalization was observed in thirty MEDIA-DHF patients (Figure 1). Of note, echocardiographic features will be reported in a separate dedicated manuscript.

Biomarker assessments in the METR-DHF cohort. A number of biomarkers were found to be differentially expressed on univariate analysis between METR-DHF participants with or without diastolic dysfunction (Table 2). The factors independently associated with diastolic dysfunction in the METR-DHF cohort are presented in Figure 1 (panel A, clinical factors: older age, higher heart rate, blood pressure and hypertension, beta-blockers and loop diuretics use; panel B: clinical factors and biomarkers). In multivariate analyses, four biomarkers were found associated with diastolic dysfunction, independently of the aforementioned clinical factors: PICP per 50 ng/ml increment (HR 1.50 (0.44 - 0.63) r=0.016), NT-proBNP > 57 pg/ml (HR 2.46 (1.27 - 2.63), p=0.008), E-selectin per 10 ng/ml (HR 0.72 (0.17 - 0.22), r=0.010), and ICAM-3 > 0.32 ng/ml (HR 3.08 (1.35 - 2.41), pO.OOOl). Their added diagnostic value in the multivariate model, as assessed by the IDI, was +1.7%, r=0.013 for PICP, +1.2%, r=0.018 for NT-proBNP, +2.1%, p=0.0006 for E-selectin, and +4.2%, pO.OOOl for ICAM-3.

Biomarker assessment in the MEDIA-DHF cohort

As predefined above, the prognostic value of these 4 biomarkers was then tested within the MEDIA-DHF cohort.

Only plasma NT-proBNP and ICAM-3 were found associated with the primary outcome on univariate analysis (Figure 2). NT-proBNP > 300 pg/ml (HR 13.5 [1.82 - 100], p=0.0l l) and ICAM-3 > 150 ng/ml (HR 3.01 [1.14 - 7.95], p=0.026) were also found associated with the primary outcome in a multivariate model (which also selected age and diabetes), while PICP > 100 ng/ml (HR 0.83 [0.40 - 1.75], p=0.63), and E-selectin > 20 ng/ml (1.37 [0.63 - 3.00], p=0.43) were not.

Mechanistics insights

In human myocardium, ICAM-3 was expressed by cardiac fibroblasts, vascular smooth muscle cells and macrophages (Example 2 and Figure 3 A). Adult human cardiac fibroblasts spontaneously expressed ICAM-3 (Figure 5). When treated with recombinant ICAM-3, they presented an increase in collagen type I secretion (p<0.0l), fibronectin expression (p<0.0l), MMP-l secretion (p<0.05) and IL-6 secretion (p<0.05) and a decrease in TIMP-l secretion (p<0.05) (Example 2 and Figure 3B). Angiotensin II and Aldosterone treatment increased ICAM-3, collagen type I, fibronectin, MMP-l, TIMP-l and IL-6 expression in control cells, whereas all these effects were blunted by ICAM-3 silencing (Example 2 and Figure 3C-D).

Discussion

This reverse translational research program provides important insights into the pathophysiology of diastolic dysfunction, which may lead to HFpEF and its dire outcomes. We found that, independent from a well-established biomarker of myocardial stress (NT-proBNP), ICAM-3 blood concentration is associated with diastolic dysfunction in patients with diastolic dysfunction at risk of HF, and is also predictive of CV outcomes in patients with symptomatic HFpEF. In addition, our in vitro experiments suggest that ICAM-3 may represent a relevant therapeutic target, since it is associated with mechanisms of fibrosis and mediates the fibrotic effects of angiotensin II and of aldosterone.

The present results fit in the current conceptual framework suggesting that myocardial remodeling and dysfunction in HFpEF results from a sequence of events consisting of the following: 1) comorbidities and especially obesity induced systemic proinflammatory state; 2) because of this proinflammatory state, coronary microvascular endothelial cells produce reactive oxygen species (ROS), which limits nitric oxide (NO) bioavailability for adjacent cardiomyocytes; 3) limited NO bioavailability decreases protein kinase G (PKG) activity in cardiomyocytes; 4) low PKG activity removes the brake on cardiomyocyte hypertrophy, thereby inducing

Concentric LV remodeling, and stiffens the cardiomyocyte due to hypophosphorylation of the giant cytoskeletal protein titin; and 5) both stiff cardiomyocytes and increased collagen deposition by myofibroblasts cause diastolic LV dysfunction, the major cardiac functional deficit in HFpEF (8). The METR-DHF results moreover show that beyond clinical features, myocardial stress (as assessed by NT-proBNP (9)), myocardial fibrosis (as assessed herein by collagen type I telopeptide -PICP (13)), and endothelial activation (as assessed by ICAM-3 and E-selectin) are associated with diastolic dysfunction, thus representing potential therapeutic targets. Surprisingly, both decreased plasma concentrations of E-selectin and increased ICAM- 3 concentrations were found associated with diastolic dysfunction, the latter exhibiting a far stronger association (Odds Ratio > 3 vs. 0.7, respectively). Furthermore, among the four aforementioned biomarkers selected as a result of the METR-DHF derivation assessment, NT- proBNP and ICAM-3 were the only biomarkers found to be associated with poor outcomes in the replication MEDIA-DHF cohort. Altogether, these clinical findings further strengthen data from the EU FP7 MEDIA consortium, showing that myocardial expression levels of the adhesion molecules E-selectin and ICAM-l were upregulated in HFpEF patients and in ZSF1- HFpEF rats, contributing to cardiac inflammation and leading to cardiac hypertrophy (18). Moreover, it has been proposed that cardiac inflammation leads to accumulation of extracellular matrix molecules contributing to diastolic dysfunction in HFpEF (19). Interestingly, biomarkers of inflammation have been found to predict severity and prognosis in HFpEF patients (20). In the present study, ICAM-3 expression by human myocardial fibroblasts was up-regulated with pro-fibrotic stimuli (aldosterone, angiotensin II) and mediated their pro-fibrotic and pro- inflammatory effects. Similarly, ICAM-l expression increases with pro-fibrotic stimuli in cardiac fibroblasts and plays a role in monocyte recruitment (21). Thus, ICAM-3 could emerge as a new therapeutic target in the context of inflammation and extracellular matrix remodeling in HFpEF.

NT-ProBNP was found associated with diastolic dysfunction (in the METR-DHF cohort) and with clinical outcomes in the MEDIA-DHF cohort. In a smaller survey (n= 86 patients), but with a longer follow-up period (median follow-up time of 579 days) and in which a proteomic (93 proteins: Proseek Multiplex96><96 CVD I vl; Olink Bioscience, Uppsala, Sweden plus NT-proBNP) strategy was implemented, 21 of the biomarkers, including several inflammatory markers outperformed NT-proBNP determined by ELISA. These markers tended to be associated with the outcome of heart failure hospitalization or death from any cause (as opposed to cardiovascular death considered in the present study). However, ICAM-3 was not assessed in this latter study (20). Notwithstanding the above, it has been shown that, compared to heart failure with reduced ejection fraction (HFrEF), HFpEF patients display lower NT- proBNP concentrations. This lower concentration of N-terminal pro-BNP is explained by concentric LV remodeling/ hypertrophy in HFpEF in contrast to eccentric LV remodeling/hypertrophy in HFrEF as well as by visceral distribution of adipose tissue in the mostly overweight or obese HFpEF patients, which is associated with decreased production and increased clearance of natriuretic peptides (9). In the METR-DHF cohort, a small rise in NT- proBNP > 57 pg/ml was already found associated with diastolic dysfunction (HR 2.46 (1.27 - 2.63), p=0.008).

In conclusion, our stepped strategy of a derivation followed by a prospective replication clinical studies, followed by in vitro mechanistic studies, provide robust evidence for microvascular endothelial activation in the development of diastolic dysfunction and HFpEF, beyond myocardial stress, thus opening promising new research avenues for a biomarker- guided therapeutic approach. Given its demonstrated association with diastolic dysfunction and outcomes in HFpEF, and owing to our mechanistic in vitro studies, ICAM-3 may represent a relevant therapeutic target and potentially enable a precision medicine approach in patients presenting isolated diastolic dysfunction or overt HFpEF.

Table 1: Baseline characteristics of the METR-DHF and MEDIA-DHF cohorts

Values are expressed as N: total number, m+SD: mean and standard deviation, n (%): number and percentage, Q2 (Q1-Q3): median (l^st-3^rd quartiles); p-values: *p<0.05, t p <0.01,

$ p<0.001.

ADHF: Acute decompensated heart failure; HF: heart failure; 1)1): diastolic dysfunction; BP: blood pressure; BMP. body mass index; eGFR: estimated glomerular filtration rate (CKD-EPI formula²²); metabolic syndrome ( Alberti 2009); discharge: from hospitalization for acute decompensation of heart failure; ACEI: angiotensin converting enzyme inhibitors; ARB: angiotensin II receptor blockers; DHP: dihydropyridines; K-sparing: potassium- sparing; PICP: Procollagen I carboxyterminal propeptide (aka CICP); NT-proBNP: N -terminal pro-B-type natriuretic peptide; ICAM-3: intercellular adhesion molecule 3.

* Due to sample volume constraints in METR-DHF, the 4 biomarkers selected for further testing in the MEDIA-DHF were possibly, measured with other methods, except for NT-proBNP. Thus their concentrations cannot be compared

Table 2. Baseline biomarkers in the METR-DHF cohort

TAC (mihoI) 259 105 (90-131 ) 14 97 (77-101 ) 0.021

Oxidized LDL (mlll/l) 259 5.58 (3.73-8.63) 14 4.33 (2.67-5.13) 0.015

Glut. R^ase (nmol/min/ml) 1 00 19.4 (12.1 -26.7) 78 1 1 .5 (9.6-12.1 ) < 0.0001

H₂O₂ (mihoI) 1 00 87.7 (68.4-104.2) 80 71 .0 (61 .8-91 .9) 0.007

P-values were obtained from the Mann- Whitney test; PIIINP: procollagen III amino- terminal peptide; MMP: matrix metalloproteinase; ICTP: I collagen telopeptide; PICP: procollagen I carboxyterminal peptide; NGAF: neutrophil gelatinase-associated lipocalin; CRP: C-reactive protein; TNFa: tumor necrosis factor alpha; s-ST2 receptor: soluble ST2 receptor; MCP-l : monocyte chemoattractant protein 1; NT-proBNP: N-terminal proBNP; GDF-15: growth differentiation factor 15; ICAM-3: intercellular adhesion molecule 3; TAC: total antioxidant capacity; FDF: low-density lipoprotein; Glut. R^ase: glutathion reductase; H2O2: hydrogen peroxyde.

EXAMPLE 2:

Material & Methods

Immunohistological evaluation

Myocardial biopsies from subjects who died from non-cardio vascular-related diseases were obtained at autopsy (n=l3). Informed consent was obtained from each subject and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution’s human research committee. Paraffin-embedded human myocardial sections (5 pm) were used. Slides were treated with H2O2 for 10 min to block peroxidase activity. All samples were blocked with 5% normal goat serum in PBS for 1 h and double staining for ICAM-3 (Abeam; dilution 1/200) and a- smooth muscle actin (Sigma; dilution 1/500) or ICAM-3 and CD68 (Abeam; dilution 1/500) was performed. Then, slides were washed three times and incubated for 30 min with the horseradish peroxidase-labeled polymer conjugated to secondary antibodies (Dako Cytomation). The signal was revealed by using DAB Substrate Kit (BD Pharmingen). As negative controls, samples followed the same procedure described above but in the absence of primary antibodies.

Cell culture

Adult human cardiac fibroblasts (HCFs) and adult human cardiomyocytes (CMs) were obtained from Promocell. Valvular interstitial cells (VICs) and valvular endothelial cells (VECs) were obtained from human aortic valves (Sadaba, JAHA 2016). All assays in the present study were done at temperatures of 37°C, 95% sterile air and 5% CO2 in a saturation humidified incubator. HCFs were used between passages 4 and 6. For experiments, HCFs were seeded into 6-well plates at 90% confluence and serum starved for l2h. Then, HCFs were cultured in the same medium and stimulated with recombinant ICAM-3 (rICAM-3; 10 ⁷ M, Abeam), Ang II (10 ⁷ M, Sigma) or Aldosterone (l0 ⁸ M, Sigma) for 6h for mRNA determinations and for 24h for protein analysis.

Transfection of cells with siRNA

Cells were seeded into 6-well plates at 70% confluence and transfected with a pool of three siRNAs (GeneCust) ICAM-3 target-specific and using MATra-si (IBA) according to the manufacturer's recommendations. Cells were allowed to recover for 24h before stimulation. Scramble siRNAs were used as a control.

Western blot analysis

Aliquots of 20 pg of total proteins were prepared from cell extracts, electrophoresed on SDS polyacrylamide gels and transferred to Hybond-c Extra nitrocellulose membranes (Bio Rad). Membranes were incubated with primary antibodies for: fibronectin (Millipore; dilution 1/1000) and ICAM-3 (Abeam; dilution 1/1000). After washing, detection was made through incubation with peroxidase-conjugated secondary antibody, and developed using an ECL chemiluminescence kit (Bio Rad). Western Blots were performed with Stain Free gels for loading control. Results are expressed as an n-fold increase over the values of the controls in densitometric arbitrary units. All Western Blots were performed at least in triplicate for each experimental condition.

ELISA

Collagen type I, MMP-l, TIMP-l and IL-6 were measured in the supernatants of the cells by ELISA according to the manufacturer's instructions (R&D Systems).

Statistical analyses

In vitro data are expressed as mean ± SEM. Normality of distributions was verified by means of the Kolmogorov-Smimov test. Data were analysed using an unpaired t test or a one way analysis of variance, followed by a Newman-Keuls to assess specific differences among groups or conditions using GraphPad Software Inc. The predetermined significance level was P < 0.05.

Results

ICAM-3 expression in myocardium

ICAM-3 expression co-localized with a-SMA positive cells as well as with cd68 positive cells indicating that ICAM-3 was expressed by cardiac fibroblasts, vascular smooth muscle cells and macrophages (Figure 3A). Moreover, cardiac fibroblasts expressed higher levels of ICAM-3 mRNA as compared to cardiac myocytes, valvular interstitial cells and valvular endothelial cells (Figure 5).

ICAM-3 induces fibrosis and inflammation in vitro

Adult human cardiac fibroblasts treated with rICAM-3 presented an increase in collagen type I secretion (p<0.0l), fibronectin expression (r<0.01), MMP-l secretion (p<0.05) and IL- 6 secretion (p<0.05) (Figure 3B). Complementary, rIC AM-3 -treated cells exhibited decreased TIMP-l secretion (p<0.05) (Figure 3B).

ICAM-3 mediates fibrosis and inflammation in vitro

In order to investigate whether ICAM-3 could be a mediator of pro-fibrotic and pro- inflammatory effects in cardiac fibroblasts, ICAM-3 -silenced cells were generated. ICAM-3 knock-down cells presented reduced ICAM-3 protein levels (70%; p<0.0l) and mRNA levels (95%; p<0.0l) (Data not shown). Angiotensin II and Aldosterone were used as known inductors of fibrosis and inflammation in human cardiac fibroblasts. Angiotensin II treatment increased ICAM-3, collagen type I, fibronectin, MMP-l, TIMP-l and IL-6 expression in control cells, whereas all these effects were blunted by ICAM-3 silencing (Figure 3C). Aldosterone also increased ICAM-3, collagen type I, fibronectin, MMP-l, TIMP-l and IL-6 expression in control cells, although ICAM-3 -knock-down cells were protected against Aldosterone effects (Figure 3D).

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of treating heart failure with preserved ejection fraction in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an ICAM-3 inhibitor.

2. The method of claim 1, wherein the ICAM-3 inhibitor is selected from the group consisting of small organic molecule, polypeptide, aptamer, antibody, oligonucleotide and ribozyme.

3. A method of identifying a patient having or at risk of having or developing heart failure with preserved ejection fraction comprising the steps of: i) determining the level of ICAM-3 and the level of NT-proBNP in a blood sample obtained from the patient, ii) comparing the level determined at step i) with a reference value 1 for ICAM-3 and a reference value 2 for a NT-proBNP, wherein detecting differential in the level of ICAM- 3 and in the level of NT-proBNP between the blood sample and the reference value 1 and the reference 2 is indicative of patient having or at risk of having or developing HFpEF.

4. A method of predicting the outcome of a patient suffering from heart failure with preserved ejection fraction comprising the steps of: i) determining the level of ICAM-3 and the level of NT-proBNP in a blood sample obtained from the patient, ii) comparing the level determined at step i) with a predetermined reference value 1 for ICAM-3 and a predetermined reference value 2 for NT-proBNP and iii) detecting differential between the levels determined at step i) with the predetermined reference value 1 and the predetermined reference value 2 indicates the outcome of the patient.

5. The method of claim 4 for predicting cardiovascular hospitalization of the patient.

6. The method of claim 4 for predicting the outcome which is cardiovascular death of the patient.

7. The method of claims 3 to 6 wherein the level of ICAM-3 and the level of NT-proBNP are determined by ELISA

8. The method of claims 4 to 7 wherein it is concluded that the patient is at risk of dying or being hospitalized when the levels determined at step i) are higher than the predetermined reference value 1 and the predetermined reference value 2.

The method of claims 4 to 8 wherein when it is concluded that the patient is at risk of dying or being hospitalized, the patient is administered with a drug selected from the group consisting of diuretics, angiotensin converting enzyme (ACE) inhibitors, digoxin (also called digitalis), calcium channel blockers, and beta-blockers.

10. The method of claims 4 to 8 wherein when it is concluded that the patient is at risk of dying or being hospitalized, the patient is administered with an ICAM-3 inhibitor.