Prognosis and treatment methods for heart failure with preserved ejection fraction using urinary protein levels.

Abstract

This specification describes methods for the prognosis and treatment of subjects with preserved ejection fraction (HFpEF) based on urinary protein levels. Such methods may include obtaining a urine sample from a subject (e.g., an HFpEF patient), measuring the urinary protein level in the sample, analyzing the urinary protein level, for example identifying elevated levels, determining, based on the analysis, whether the subject is at risk of death or hospitalization for heart failure (DHFA), and further including treating the subject based on the risk determination.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application Nos. 63 / 485,770 and 63 / 488,158, filed on February 17, 2023 and March 2, 2023, respectively, and the entire contents of each of these provisional patent applications are incorporated herein by reference.

[0002] The present invention relates to methods for predicting the prognosis and treating a subject with heart failure (HF) with preserved ejection fraction (HFpEF) based on urinary protein levels.

Background Art

[0003] Heart failure with preserved ejection fraction (HFpEF) accounts for approximately half of all HF diagnoses. There is no established drug therapy to reduce the all - cause mortality of HFpEF, and the therapeutic options for reducing the risk of cardiovascular death or hospitalization are limited. There is a need for a prognostic method for HFpEF to identify subjects at risk of cardiovascular death or hospitalization. Such a prognosis may enable medical and therapeutic interventions to reduce the risk of death or hospitalization.

[0004] Urinary protein / peptide (UPP) is non - invasive, inexpensive to collect, and usually has no side effects or complications. Notably, the urine proteome includes information not only from the kidney and urinary tract but also from other organs due to glomerular filtration of some plasma proteins. Urinary protein / peptide has the potential as a disease biomarker.

Summary of the Invention

[0005] This specification describes methods for the prognosis and treatment of subjects with preserved ejection fraction (HFpEF) based on urinary protein levels. Such methods may include obtaining a urine sample from a subject (e.g., an HFpEF patient), measuring the urinary protein level in the sample, analyzing the urinary protein level, for example identifying elevated levels, determining, based on the analysis, whether the subject is at risk of death or hospitalization for heart failure (DHFA), and further including treating the subject based on the risk determination. [Brief explanation of the drawing]

[0006] [Figure 1A] This is a volcano plot showing the standardized hazard ratios for urinary protein associated with the composite outcome of death or hospitalization for heart failure (DHFA) in the unadjusted analysis. The dashed lines represent the unadjusted (lower) and adjusted (upper) significance levels. [Figure 1B] This is a volcano plot showing the standardized hazard ratios for urinary protein associated with the composite outcome of death or hospitalization for heart failure (DHFA), after adjusting for urinary creatinine and MAGGIC scores. The dashed lines represent the unadjusted (lower) and adjusted (upper) significance levels. [Modes for carrying out the invention]

[0007] As mentioned above, a prognostic method for HFpEF is needed for risk stratification, including identifying subjects at risk of death or hospitalization for heart failure (DHFA) and treating them based on their prognosis. Previous proteomics studies in HFpEF have focused on proteins in plasma. Studies on urinary proteomics in HFpEF are rare (see, e.g., He T, et al. European journal of heart failure. 2021;23(11):1875-87), and there has been no previously available data on prognostic values ​​of urinary proteomics in HFpEF.

[0008] Currently, certain urinary proteins are known to have prognostic values ​​in risk stratification of HFpEF. For example, certain urinary proteins can be used to determine the risk of DHFA. This disclosure provides a prognostic method for determining the severity of HFpEF, which includes determining the risk of DHFA in a subject by measuring and / or analyzing the levels of one or more urinary proteins in the subject. These and other prognostic methods, as well as various embodiments thereof, are described in further detail below.

[0009] Prognosis and risk stratification based on urinary protein levels can be applied to methods of treating subjects, such as HFpEF patients. Prognosis can be determined periodically before, after, and / or during treatment, and the course of treatment can be determined using prognosis and / or changes in prognosis. For example, subjects at high risk for DHFA may receive more invasive treatment or different therapies than subjects at low risk for DHFA. These and other treatment methods, as well as their various embodiments, are described in further detail below.

[0010] definition Various embodiments and aspects of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments and aspects are provided only as examples. Numerous variations, modifications, and substitutions will be conceivable to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention.

[0011] Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Any methods, apparatus, and materials similar or equivalent to those described herein may be used in the practice of the present invention.

[0012] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books, manuals and articles, are expressly incorporated herein by reference in whole for any purpose.

[0013] The terms "a" or "an" as used herein mean one or more.

[0014] The terms “comprise,” “include,” “have,” and their derivatives are used interchangeably herein as comprehensive and unrestricted terms. For example, the use of “comprising,” “including,” or “have” means that any element that constitutes, has, or is included is not the only element contained within the subject of the clause containing the verb.

[0015] Where used herein, “treatment,” “to treat,” or “to improve” are interchangeable herein. These terms refer to methods for obtaining beneficial or desired outcomes, including but not limited to therapeutic benefits. Therapeutic benefits mean the eradication or improvement of an underlying disease being treated, and / or the eradication or improvement of one or more physiological symptoms associated with the underlying disease, even though the subject may still be suffering from the underlying disease. Treatment includes delaying the onset of clinical symptoms of a disease by administration of a composition; suppression of the disease, i.e., causing a reduction in the clinical symptoms of the disease; inhibition of the disease, i.e., preventing the onset of clinical symptoms by administration of a composition after the initial appearance of symptoms; and / or mitigation of the disease, i.e., causing a regression of clinical symptoms by administration of a composition after the initial appearance of symptoms.

[0016] "Patient" or "Subject" means an organism that is suffering from or susceptible to a disease or condition that can be treated using the methods provided herein. The term does not necessarily imply that the subject has been diagnosed with a specific disease, but typically refers to an individual under medical care. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, cats, monkeys, goats, sheep, deer, and other non-mammals. In some embodiments, the patient or subject is human. In some embodiments, the patient or subject is suffering from or has been diagnosed with HFpEF.

[0017] Embodiment This disclosure provides a method for prognosing HFpEF based on urinary protein levels. In one aspect, the Spec. provides a method for determining the risk of death or hospitalization for heart failure (DHFA) in a subject, comprising measuring or having measured one or more urinary protein levels in the subject's urine, analyzing or having analyzed one or more urinary protein levels, and determining the risk of DHFA in the subject based on the analysis of one or more urinary protein levels.

[0018] This disclosure also provides a method for treating HFpEF in conjunction with the prognosis of the subject. In one embodiment, this specification provides a method for treating heart failure with preserved ejection fraction (HFpEF) in a subject in need, the method comprising measuring or having measured one or more levels of urinary protein in the subject's urine, analyzing or having analyzed one or more levels of urinary protein, determining or having determined the risk of death or hospitalization for heart failure (DHFA) in the subject based on the analysis of one or more levels of urinary protein, and treating the subject with HFpEF therapy. In some embodiments, if the subject is determined to be at risk for DHFA, a first HFpEF therapy is administered, and if the subject is determined to be at no risk for DHFA, a second HFpEF therapy is administered. In some embodiments, if the subject is determined to be at high risk for DHFA, a first HFpEF therapy is administered, and if the subject is determined to be at low risk for DHFA, a second HFpEF therapy is administered. In some embodiments, the first HFpEF therapy and the second HFpEF therapy each involve the administration of the same therapeutic agent, wherein the first HFpEF therapy involves the administration of a higher dose of the therapeutic agent compared to the second HFpEF therapy. In some embodiments, the first HFpEF therapy involves the administration of a different therapeutic agent than that of the second HFpEF therapy.

[0019] In some embodiments, the first HFpEF therapy and / or the second HFpEF therapy is a guideline-based pharmacotherapy (GDMT) for HFpEF or associated comorbidities, such as hypertension and coronary artery disease. In some embodiments, the first HFpEF therapy and / or the second HFpEF therapy is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), SGLT2 inhibitors, diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine, or any combination thereof. In some embodiments, the beta-blockers are selected from the group consisting of bisoprolol, carvedilol, carvedilol, and metoprolol succinate, and include their controlled-release and long-release forms as well as their pharmaceutically acceptable salts. In some embodiments, the ACE inhibitor is selected from the group consisting of captopril, enalapril, hosinopril, lisinopril, perindopril, quinapril, ramipril, and trandolapril, and their pharmaceutically acceptable salts. In some embodiments, the ARB is selected from the group consisting of candesartan, losartan, and valsartan, and their pharmaceutically acceptable salts. In some embodiments, the ARN inhibitor is sacubitril-valsartan. In some embodiments, the MRA is spironolactone or eplerenone, or their pharmaceutically acceptable salts. In some embodiments, the soluble guanylate cyclase stimulant is vericiguat or digoxin, or their pharmaceutically acceptable salts. In some embodiments, the diuretic is selected from the group consisting of bumetanide, furosemide, torasemide, chlorothiazide, chlorthalidone, hydrochlorothiazide, indapamide, and metrazone, or their pharmaceutically acceptable salts. In some embodiments, the diuretic is selected from the group consisting of bumetanide, furosemide, and torasemide, or pharmaceutically acceptable salts thereof.In some embodiments, the GLP-1 agonist is selected from the group consisting of tylzepatide, semaglutide, lixisenatide, lilaglutide, exenatide, and dulaglutide, or pharmaceutically acceptable salts thereof. In some embodiments, the GLP-1 agonist is tylzepatide or semaglutide, or a pharmaceutically acceptable salt thereof. In some embodiments, the calcium sensitizer is levocimendan or a pharmaceutically acceptable salt thereof.

[0020] In some embodiments, the second HFpEF therapy is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, and mineralocorticoid receptor antagonists (MRAs). In some embodiments, the second HFpEF therapy is selected from the group consisting of beta-blockers, angiotensin receptor blockers (ARBs), and mineralocorticoid receptor antagonists (MRAs). In some embodiments, the second HFpEF therapy is a beta-blocker.

[0021] In some embodiments, the first HFpEF therapy is (a) a pharmacotherapy based on different guidelines for HFpEF than the second HFpEF therapy, or (b) a pharmacotherapy based on the same guidelines but at a different dose (e.g., a higher dose) than the second HFpEF therapy. In some embodiments, the first HFpEF therapy may be selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), SGLT2 inhibitors, diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine. In some embodiments, the first HFpEF therapy is a more invasive therapy. In some embodiments, the first HFpEF therapy is a higher dose of the second HFpEF therapy. In some embodiments, the first HFpEF therapy is an SGLT2 inhibitor. In some embodiments, the SGLT2 inhibitor is dapagliflozin or a pharmaceutically acceptable salt thereof. In some embodiments, the SGLT2 inhibitor is empagliflozin or a pharmaceutically acceptable salt thereof. In some embodiments, the first HFpEF therapy is aficumten or a pharmaceutically acceptable salt thereof.

[0022] In some embodiments, the first HFpEF therapy includes the administration of several therapeutic agents, such as those described above. For example, the first HFpEF therapy may include a beta-blocker and an SGLT2 inhibitor. The first HFpEF therapy may include a beta-blocker and an ARB, a beta-blocker and an MRA, an ARB and an MRA, or all three of the beta-blocker, ARB and an MRA.

[0023] In some embodiments, the first HFpEF therapy may include administration of a therapeutic agent (e.g., as described above) at a dose greater than the corresponding dose of the therapeutic agent by the second HFpEF therapy. For example, the first HFpEF therapy dose may be 1.5 times or more, 2 times or more, 3 times or more, or 4 times or more the dose by the second HFpEF therapy. For example, the first HFpEF therapy dose may be more than 1.5 to 2 times, 1.5 to 3 times, 2 to 3 times, 2 to 4 times, 3 to 5 times or 4 to 5 times the dose by the second HFpEF therapy.

[0024] More generally, urinary protein levels can be used for prognostic and risk stratification of HFpEF. In another aspect, provided herein is a method for assessing the severity of HFpEF in a subject, comprising measuring or having measured one or more urinary protein levels in the urine of the subject, analyzing or having analyzed the one or more urinary protein levels, and determining the severity of HFpEF in the subject based on the analysis of the one or more urinary protein levels. In some embodiments, the severity of HFpEF is characterized by the risk of death or hospitalization for heart failure (DHFA) of the subject.

[0025] The method also includes, for example, monitoring the prognosis of the subject to track the subject's condition over time. Such monitoring can be useful in determining whether to maintain or change the course of treatment of the subject. In another aspect, provided herein is a method for monitoring the severity of HFpEF in a subject who has HFpEF or has been diagnosed with HFpEF, comprising measuring or having measured one or more urinary protein levels in the urine of the patient, analyzing or having analyzed the one or more urinary protein levels, and determining the severity of HFpEF in the patient based on the analysis of the one or more urinary protein levels. In some embodiments, the severity of HFpEF is characterized by the risk of death or hospitalization for heart failure (DHFA) of the subject.

[0026] It is contemplated that a particular drug(s) can be selected based on the specific urinary protein(s) measured and analyzed, and the particular drug(s) is known to have a functional effect on the urinary protein(s). In another aspect, provided herein is a method for identifying treatment options for a subject at risk of death or heart failure hospitalization (DHFA), the method comprising: (a) obtaining or having obtained a urine sample from the subject; (b) measuring or having measured one or more urinary protein levels in the sample; (c) analyzing or having analyzed the urinary protein levels in the sample; (d) selecting at least one urinary protein based on the analysis; and (e) selecting a drug or a combination of drugs that has a functional effect on at least one urinary protein selected in (d). In some embodiments, the method further comprises (f) administering the drug or combination of drugs to the subject. In some embodiments, the method further comprises (g) measuring at least one urinary protein selected in (d) after administration of the drug or combination of drugs to determine whether the urinary protein level has changed, for example, by determining that the urinary protein level is no longer increasing.

[0027] A more general method of risk stratification is applicable to the treatment of HFpEF. In another aspect, provided herein is a method for treating heart failure with preserved ejection fraction (HFpEF) in a subject in which the ejection fraction is maintained, the method comprising measuring or having measured one or more urinary protein levels in the urine of the subject, analyzing or having analyzed the one or more urinary protein levels, determining or having determined the severity of HFpEF in the subject based on the analysis of the one or more urinary protein levels, and treating the subject with HFpEF therapy.

[0028] In another aspect, the Specified provides a method for determining the risk of death or hospitalization for heart failure (DHFA) in a subject, comprising determining the risk of DHFA in the subject based on one or more urinary protein levels.

[0029] As described above, a more invasive, less invasive, or alternative therapy may be employed based on the prognosis of the subject. In another embodiment, this specification provides a method for treating heart failure with preserved ejection fraction (HFpEF) in a subject in need, comprising determining one or more levels of urinary protein in the subject, administering a first HFpEF therapy to the patient if one or more levels of urinary protein are above a threshold, and administering a second HFpEF therapy to the patient if one or more levels of urinary protein are below a threshold. In some embodiments, the second HFpEF therapy comprises a different drug from the first therapy. In some embodiments, the first HFpEF therapy comprises a first drug in a first dosage, and the second therapy comprises a first drug in a second dosage.

[0030] In another embodiment, this specification provides a method for treating heart failure with preserved ejection fraction (HFpEF) in a subject in need, wherein the subject is given a first HFpEF therapy, the method comprises determining one or more urinary protein levels in the subject, and if one or more urinary protein levels are above a threshold, the patient is given a second HFpEF therapy. In some embodiments, the second HFpEF therapy comprises a different drug from the first therapy. In some embodiments, the first HFpEF therapy comprises a first drug in a first dose, and the second therapy comprises a first drug in a second dose.

[0031] In another embodiment, this specification provides a method for treating heart failure with preserved ejection fraction (HFpEF) in a subject in need, comprising administering a first HFpEF therapy to the subject, determining one or more urinary protein levels in the subject, and if one or more urinary protein levels are below a threshold, administering a second HFpEF therapy to the patient. In some embodiments, the second HFpEF therapy comprises a different drug from the first therapy. In some embodiments, the first HFpEF therapy comprises a first drug in a first dose, and the second therapy comprises a first drug in a second dose.

[0032] In these and other embodiments, the method can be carried out according to various embodiments, as described below, for example.

[0033] In some embodiments, the subject has and / or has been diagnosed with heart failure with preserved ejection fraction (HFpEF). In some embodiments, the HFpEF is high-contractile HFpEF. In some embodiments, the HFpEF is low-contractile HFpEF. In some embodiments, the subject has and / or has been diagnosed with symptomatic heart failure. In some embodiments, the subject has and / or has been diagnosed with NYHA class II, III, or IV heart failure. In some embodiments, the subject has symptomatic heart failure and a left ventricular ejection fraction (LVEF) of 45% or greater. In some embodiments, the subject has symptomatic heart failure and a left ventricular ejection fraction (LVEF) of 50% or greater.

[0034] In some embodiments of this method, subjects are determined to be at risk of HFpEF before the onset of HFpEF. In some embodiments, subjects are considered to be at risk of HFpEF because they have and / or have been diagnosed with hypertrophic cardiomyopathy (HCM). In some embodiments, subjects are considered to be at risk of HFpEF because they have and / or have been diagnosed with NYHA class I heart failure.

[0035] In some embodiments of this method, the subjects are HFpEF patients who are resistant to or intolerant to guideline-based drug therapy.

[0036] This method may include one or more of the following: measuring one or more urinary protein levels, analyzing one or more urinary protein levels, and determining the prognosis of the subject (e.g., determining the risk of DHFA).

[0037] Analyzing protein levels may include comparing urinary protein levels to a threshold to determine if protein levels are elevated. If multiple protein levels are measured, analyzing protein levels may include calculating a composite score based on the protein levels and comparing the composite score to a threshold. If multiple protein levels are measured, analyzing protein levels may include analyzing whether multiple, majority, or specific percentages (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of protein levels are elevated. Analyzing protein levels may also include determining the extent to which protein levels are elevated compared to normal or threshold levels (e.g., a percentage above normal).

[0038] Determining the prognosis (e.g., DHFA risk) may involve referring to the correlation between elevated levels of protein(s) and the prognosis (e.g., DHFA risk) in order to reach the subject's prognosis. Determining the prognosis may further involve communicating or outputting the subject's prognosis.

[0039] Measuring one or more urinary protein levels may include measuring proteins in a urine sample by liquid chromatography-mass spectrometry (LC-MS). Other suitable methods known in the art for measuring urinary protein levels, such as other mass spectrometry and immunoassays, may be employed. See, for example, Aitekenov S, et al., Review: Detection and quantification of proteins in human urine. Talanta. 2021 Feb 1;223(Pt 1):121718.

[0040] In some embodiments, one or more urinary protein levels include the levels of one or more proteins selected from the group consisting of MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, haptoglobin, COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868 (including combinations thereof). These 40 elevated protein levels were identified in the Example 1 study as having a direct or inverse correlation with DHFA results in HFpEF subjects in unadjusted analysis. In some embodiments, one or more urinary protein levels include the levels of one or more proteins selected from the group consisting of DNASE1, AMY2A, ANGPTL2, EGF, ZG16B, ALDH1A1, ICOSLG, PLAU, HSP60, AMY2B, B4GALT1, SLC2A5, and COL15A1 (including combinations thereof).

[0041] In some embodiments, one or more proteins include glutathione S-transferase Mu3. Elevated levels of this protein were identified as correlated with DHFA outcomes in analyses using a urinary creatinine-adjusted model, similar to how elevated levels of this protein correlate with DHFA outcomes in HFpEF subjects.

[0042] In some embodiments, one or more urinary protein levels include levels of one or more proteins selected from the group consisting of MBD1, IGHA2, ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868 (including combinations thereof). These 21 elevated levels of proteins were identified in the Example 1 study as having direct or inverse correlations with DHFA outcomes in HFpEF subjects in a model adjusted for urinary creatinine levels and the Meta-Analysis Global Group (MAGGIC) risk score for chronic HF. In some embodiments, one or more urinary protein levels include the levels of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, AMY2B, PLAU, HSP60, ZG16B, ICOSLG, and PGKA (including combinations thereof).

[0043] In some embodiments, measured and / or analyzed urinary protein levels are directly correlated with increased severity of HFpEF and / or DHFA risk. In some embodiments, one or more urinary protein levels include levels of one or more proteins selected from the group consisting of MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, and haptoglobin (including combinations thereof). These eight proteins were found to be directly correlated with DHFA risk in unadjusted analysis. In some embodiments, one or more urinary protein levels include levels of one or more proteins selected from the group consisting of MBD1 and IGHA2 (including combinations thereof). These two proteins were found to be directly correlated with DHFA risk in a model adjusted for urinary creatinine levels and the Meta-Analysis Global Group (MAGGIC) risk score for chronic HF.

[0044] In some embodiments, measured and / or analyzed urinary protein levels are inversely correlated with increased severity of HFpEF and / or DHFA risk. In some embodiments, one or more urinary protein levels include levels of one or more proteins selected from the group consisting of COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868 (including combinations thereof). These 32 proteins were found to be inversely correlated with DHFA risk in unadjusted analysis. In some embodiments, one or more urinary protein levels include levels of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868 (including combinations thereof). These 19 proteins were found to be inversely correlated with DHFA risk in a model adjusted for urinary creatinine levels and the Meta-Analysis Global Group (MAGGIC) risk score for chronic HF. In some embodiments, one or more urinary protein levels include levels of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, and SLC2A5 (including combinations thereof).

[0045] In some embodiments, the measured and / or analyzed urinary protein levels are those of fibrosis-related proteins, such as collagen-derived proteins. For example, urinary proteins may include COL15A1, COL6A1, or combinations thereof, which are collagen-derived proteins involved in fibrosis.

[0046] In some embodiments, the measured and / or analyzed urinary protein levels are those of proteins involved in metabolism, such as carbohydrate metabolism or lipid metabolism. For example, urinary proteins may include AMY2A, AMY2B, MAN1A1, or combinations thereof, which are related to carbohydrate metabolism. For example, urinary proteins may include ALDH1A1, B4GALT1, or combinations thereof, which are related to lipid metabolism. In some embodiments, the measured and / or analyzed urinary protein levels are those of pancreatic alpha-amylases, such as AMY2A, AMY2B, or combinations thereof.

[0047] In some embodiments, the measured and / or analyzed urinary protein levels are those of proteins involved in inflammation. For example, the measured and / or analyzed urinary protein may include ANGPTL2, which is known to be involved in chronic inflammation. The measured and / or analyzed urinary protein may also include HSPD1 and / or ATP1B1, which are involved in inflammation.

[0048] In some embodiments, the measured and / or analyzed urinary protein levels are those of epithelial sodium channel stimulants such as prostacin.

[0049] In some embodiments, the measured and / or analyzed urinary protein levels include the level of ANGPTL2.

[0050] In some embodiments, the measured and / or analyzed urinary protein levels include DNASE1 levels.

[0051] In some embodiments, the measured and / or analyzed urinary protein levels include ALDH1A1 levels.

[0052] In some embodiments, the measured and / or analyzed urinary protein levels include the level of SLC2A5.

[0053] In some embodiments, the measured and / or analyzed urinary protein levels include AMY2A levels.

[0054] In some embodiments, the measured and / or analyzed urinary protein levels include AMY2B levels.

[0055] In some embodiments, the measured and / or analyzed urinary protein levels include the MBD1 level.

[0056] In some embodiments, the measured and / or analyzed urinary protein levels include IGHA2 levels.

[0057] In some embodiments, the measured and / or analyzed urinary protein levels include prostacin levels.

[0058] In some embodiments, analyzing one or more urinary protein levels includes determining whether one or more urinary protein levels are elevated. In some embodiments, analyzing one or more urinary protein levels includes comparing one or more urinary protein levels to a threshold. In some embodiments, determining the risk of DHFA includes determining whether one or more urinary protein levels are above or below a threshold.

[0059] In some embodiments, the method of the present disclosure further includes determining that a subject has an increased risk of DHFA if one or more urinary protein levels are elevated. In some embodiments, if the increased risk of DHFA correlates with elevated protein levels, one or more urinary protein levels include levels of one or more proteins selected from the group consisting of MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, and haptoglobin (including combinations thereof). In some embodiments, if the increased risk of DHFA correlates with elevated protein levels, one or more urinary protein levels include levels of one or more proteins selected from the group consisting of MBD1 and IGHA2 (including combinations thereof).

[0060] In some embodiments, the method of the present disclosure includes determining that a subject has a reduced risk of DHFA if one or more urinary protein levels are elevated. In some embodiments, if a reduced risk of DHFA correlates with elevated protein levels (i.e., an increased risk of DHFA is inversely correlated with elevated protein levels), then one or more urinary protein levels include the levels of one or more proteins selected from the group consisting of COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868 (including combinations thereof). In some embodiments, if a decrease in DHFA risk correlates with an increase in protein levels (i.e., if an increase in DHFA risk is inversely correlated with an increase in protein levels), one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868 (including combinations thereof). In some embodiments, if a decrease in DHFA risk correlates with an increase in protein levels (i.e., if an increase in DHFA risk is inversely correlated with an increase in protein levels), one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, and SLC2A5 (including combinations thereof).

[0061] In some embodiments, the method of the present disclosure further includes obtaining, or having obtained, a urine sample from a subject, from which one or more urinary protein levels are measured. In some embodiments, the urine sample is frozen after acquisition and before measurement of urinary protein levels.

[0062] In some embodiments, the methods of the present disclosure further include treating a subject with a certain therapy based on the risk of DHFA in the subject. In some embodiments, the therapy is HFpEF therapy.

[0063] In another embodiment, this specification provides a method for treating heart failure with preserved ejection fraction (HFpEF) by administering a therapy to a subject in need. In some embodiments, the therapy is a therapy known in the art for the treatment of HFpEF. In some embodiments, the subject is identified as being in need of treatment based on the level of one or more urinary proteins in the subject. In some embodiments, the urinary proteins are one or more urinary proteins disclosed herein, for example, in Table 2. In some embodiments, the urinary proteins are MBD1 and / or IGHA2. In some embodiments, the urinary proteins are ANGPTL2, AMY2A and / or SLC2A5. In some embodiments, the method includes one or more of the following: obtaining or having obtained a urine sample from the subject; measuring or having measured the level of urinary protein in the sample; analyzing or having analyzed the level of urinary protein in the sample against a standard or threshold level; and determining or having determined, based on the analysis, whether to treat the subject. In some embodiments, the subject is monitored during treatment to determine the need for treatment or adjustment of treatment. In some embodiments, the method includes, or has been, obtaining a urine sample from a subject receiving treatment for HFpEF; measuring or having measured the level of urinary protein in the sample; analyzing or having analyzed the level of urinary protein in the sample against a standard or threshold level; and deciding or having decided, based on the analysis, whether to discontinue, continue, or modify the subject's treatment. In some embodiments, the analysis includes quantitative analysis. In some embodiments, the analysis includes correlating the measured urinary protein level with the risk of DHFA. In some embodiments, the analysis of urinary protein(s) in the urine sample is used to determine the subject's prognosis as having a risk of death or hospitalization for heart failure (DHFA) (e.g., high risk). In some embodiments, the analysis of urinary protein(s) in the urine sample is used to determine the subject's prognosis as having no risk or low risk of death or hospitalization for heart failure (DHFA).In some embodiments, if the method includes the prognosis of a subject as being at risk of DHFA (e.g., high risk), the method further includes discontinuing, continuing, or modifying treatment based on the prognosis, and / or the method further includes initiating or monitoring other treatments to reduce the risk of DHFA.

[0064] This method may include discontinuing, continuing, or modifying treatment based on urinary protein analysis (and / or related prognosis). The discontinued, continued, or modified treatment may be a therapy ("T1") consisting of a pharmaceutically acceptable salt of a pharmaceutically acceptable salt selected from the group comprising beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), SGLT2 inhibitors, diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine. For example, if it is determined during treatment with T1 that the prognosis is deteriorating and / or that urinary protein levels are not within the target range or target level (or are not progressing toward the target range or target level), T1 may be discontinued. If, during treatment with T1, it is determined that the prognosis is improving and / or that the urinary protein level(s) are within the target range or at the target level (or progressing toward the target range or at the target level), T1 may be continued. Alternatively, if, during treatment with T1, it is determined that the prognosis is worsening or not improving and / or that the urinary protein level(s) are not within the target range or at the target level (or not progressing toward the target range or at the target level), T1 may be changed.

[0065] Modifying T1 may include discontinuing T1 and initiating a second therapy ("T2"). Modifying T1 may include continuing T1 and initiating a second therapy ("T2") together with T1. Modifying T1 may include increasing the dose of T1. Modifying T1 may include decreasing the dose of T1, with or without initiating a second therapy ("T2"). In some embodiments, T2 is a more invasive therapy than T1.

[0066] In some embodiments, T2 is an SGLT2 inhibitor. In some embodiments, T2 is afficamten or a pharmaceutically acceptable salt thereof. In some embodiments, T1 is a beta-blocker, an angiotensin receptor blocker (ARB), or a mineralocorticoid receptor antagonist (MRA), and T2 is an SGLT2 inhibitor. In some embodiments, T1 is a beta-blocker, an angiotensin receptor blocker (ARB), or a mineralocorticoid receptor antagonist (MRA), and T2 is afficamten or a pharmaceutically acceptable salt thereof.

[0067] In some embodiments, T2 is a less invasive therapy than T1, e.g., a less invasive drug. In some embodiments, modifying T1 includes reducing the dose of the T1 therapy.

[0068] In some embodiments, T2 is a different therapy from T1, comprising a pharmaceutically acceptable salt thereof, selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), SGLT2 inhibitors, diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine.

[0069] In some embodiments, modifying T1 may involve initiating a mechanical intervention, regardless of whether T1 therapy is continued or whether T2 therapy is initiated. In some embodiments, the mechanical intervention is mechanical circulatory support (MCS). In some embodiments, the mechanical intervention is an atrial shunt, a left ventricular dilator, or a nerve stimulator.

[0070] In some embodiments, T2 includes the administration of multiple therapeutic agents, for example, as described above. For example, T2 may include an SGLT2 inhibitor with one or more of the beta-blockers, ARBs, and MRAs. T2 may include afficamten or a pharmaceutically acceptable salt thereof with one or more of the beta-blockers, ARBs, and MRAs. T2 may include a beta-blocker and an ARB, a beta-blocker and an MRA, an ARB and an MRA, or all three of the beta-blocker, ARB, and MRA.

[0071] In some embodiments, T2 may involve administering a dose of the therapeutic agent (e.g., as described above) greater than the corresponding dose of the therapeutic agent administered by T1. For example, the dose of T2 may be 1.5 times, 2 times, 3 times, or 4 times or more of the dose administered by T1. For example, the dose of T2 may be 1.5 to 2 times, 1.5 to 3 times, 2 to 3 times, 2 to 4 times, 3 to 5 times, or more than 4 to 5 times the dose administered by T1.

[0072] In another embodiment, this specification provides a method for diagnosing (or prognosing) the risk of death or hospitalization for heart failure (DHFA) in a subject, comprising measuring the urinary protein level in the subject. In some embodiments, the subject is a patient with HFpEF. In some embodiments, HFpEF is high-contractile HFpEF. In some embodiments, HFpEF is low-contractile HFpEF. In some embodiments, the urinary protein is one or more urinary proteins disclosed herein, for example, in Table 2. In some embodiments, the urinary protein(s) are MBD1 and / or IGHA2. In some embodiments, the urinary protein(s) are ANGPTL2, AMY2A and SLC2A5. In some embodiments, the method comprises one or more of the following: obtaining or having obtained a urine sample from the subject; measuring or having measured the urinary protein level in the sample; analyzing or having analyzed the urinary protein level in the sample against a standard or threshold level; and diagnosing (or prognosing) or having diagnosed (or prognosed) the risk of DHFA in the subject based on the analysis. In some embodiments, if a subject has urinary protein levels above the normal range or above a threshold, the subject is determined to be at risk or high risk for DHFA. In some embodiments, if a subject has elevated levels of urinary protein selected from MBD1, IGHA2, or a combination thereof, the subject is determined to be at risk or high risk for DHFA. In some embodiments, if a subject has urinary protein levels above the normal range or above a threshold, the subject is determined to be at no risk or low risk for DHFA. In some embodiments, if a subject has elevated levels of urinary protein selected from ANGPTL2, AMY2A, and SLC2A5, or any combination thereof, the subject is determined to be at no risk or low risk for DHFA. In some embodiments, the method further includes treating the subject with a certain therapy based on the subject's risk for DHFA.

[0073] Therapies for HFpEF are described in the art, for example, in *Heart Failure with Preserved Ejection Fraction: Mechanisms and Treatment Strategies*, Kazunori Omote, Frederik H. Verbrugge, Barry A. Borlaug, *Annual Review of Medicine* 2022 73:1, 321-337, which is incorporated herein by reference in its entirety.

[0074] In another embodiment, this specification provides a test kit for diagnosing (or prognosing) the risk of DHFA in a subject, or for assessing the severity of HFpEF in a subject. In some embodiments, the kit includes a collection device for collecting and / or storing a urine sample. In some embodiments, the collection device may be a container, e.g., a sterile container. In some embodiments, the kit further includes a measuring device for qualitatively or quantitatively measuring one or more urinary protein levels in the subject's urine. In some embodiments, the measuring device may be a urinary protein test strip. In some embodiments, the kit further includes software for analyzing the measured protein levels. The software may be configured to input one or more urinary protein levels. The software may perform an analysis comparing the urinary protein level(s) to a threshold(s). The analysis may identify the urinary protein level(s) as elevated or not elevated. The software may calculate a composite score, the percentage of protein at the elevated level, and / or the degree to which the urinary protein level is elevated. The software may output the subject's prognosis with respect to the severity of HFpEF. The software may output a determination of the subject's risk of DHFA. In some embodiments, the software may output proposed therapeutic interventions based on an analysis of measured protein levels. [Examples]

[0075] Example 1 - Urinary proteomics and results in heart failure with preserved ejection fraction In this study, novel urinary protein / peptide measurements were performed on frozen urine samples available from 426 participants enrolled in the aldosterone antagonist trial for the treatment of HF with preserved cardiac function (TOPCAT). The relationship between urinary biomarker levels and the risk of death or HF-related hospitalization (DHFA) in this cohort was evaluated.

[0076] In the adjusted analysis, the top urinary proteins / peptides associated with DHFA were angiopoietin-like protein 2 (ANGPTL2) (HR=0.5731, CI=0.47-0.7, P=3.13E-05), alpha-amylase 2A (AMY2A) (HR=0.5496, CI=0.44-0.69, P=0.0001), and deoxyribonuclease-1 ( This included DNASE1 (HR=0.5704, CI=0.46-0.71, P=0.0002), aldehyde dehydrogenase 1 family member A1 (ALDH1A1) (HR=0.5572, CI=0.47-0.72, P=0.0003), and alpha-amylase 2B (AMY2B) (HR=0.5841, CI=0.44-0.7, P=0.0003). Higher urinary levels of various proteins involved in fibrosis (collagen VI alpha-1, collagen XV alpha-1), metabolism (pancreatic alpha-amylase 2A / B, mannosidase alpha class 1A member 1), and inflammation (heat shock protein family D member 1, inducible T cell costimulatory ligand) were associated with a lower risk of DHFA.

[0077] method Test subject group The individuals included in this analysis were participants in the aldosterone antagonist trial for the treatment of heart failure with preserved ejection fraction (TOPCAT). TOPCAT was a multicenter, double-blind, placebo-controlled, randomized trial of spironolactone that enrolled 3,445 adults with HFpEF (LVEF ≥ 45%) from six countries between 2006 and 2012. The trial design, characteristics, inclusion / exclusion criteria, and results have already been published. See Pitt B, Pfeffer MA, Assmann SF, Boineau R, Anand IS, Claggett B, et al. Spironolactone for heart failure with preserved ejection fraction. N Engl J Med. 2014;370:1383-92.

[0078] Urine biomarker sample Urine samples for biomarker analysis were obtained from a subset of TOPCAT trial participants who had samples available for novel proteomic analysis. Approximately 270 μl of urine from each participant was ali-coated into 96-well deep-well plates. Each plate contained four wells of pooled urine samples from normal, healthy volunteers designated as quality control samples. The urine sample plates were stored at -80°C until the start of the experiment. Each plate was prepared for proteomic analysis on a separate day. Urine proteins were reduced and alkylated by adding 30 μl of 0.1 M DTT and 0.2 M IAA, and then incubated in a thermoshaker at 60°C and 1000 rpm for 1 hour. The samples were cooled to room temperature, then mixed with 900 μl of cold acetonitrile and incubated overnight at -20°C. The plates were centrifuged in a plate centrifuge at 2500 g for 20 minutes at room temperature. The supernatant was aspirated using a 1.2 ml pipette with a multichannel pipette. The protein pellet was washed with 1 ml of 100% acetonitrile at room temperature. The plate was shaken in a plate shaker for 5 minutes, then centrifuged at 2500 g for 10 minutes at room temperature. Finally, the supernatant was carefully aspirated, leaving a clean pellet behind. The pellet was air-dried for 5 minutes. The protein pellet was dissolved in 100 μl of freshly prepared 8 M urea containing 100 mM Tris HCl. The protein was digested with 0.5 μg of LysC (Wako Chemicals; Richmond, VA) at 37°C for 4 hours at 1000 rpm. The partially digested sample was diluted with 400 μl of LCMS-grade water, and 1 μg of trypsin / LysC mix (Promega, Madison, WI) was added. This plate was incubated overnight at 37°C at 1000 rpm to completely digest the protein. Peptide concentrations were measured using tryptophan fluorescence.

[0079] LC MS / MS analysis A total of 500 ng of each sample was loaded into individual Evotip (Evosep, Denmark), washed with 50 μl of 0.1% formic acid (FA), and then 100 μl of storage solvent (0.1% FA) was added to keep the Evotip moist until analysis. The Evosep One system (Evosep) was coupled online to a QExactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA) using a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were eluted from the Evotip on a Pepsep C-18 reversed-phase column (ReproSil 3 μm, 120 Å, 8 cm 75 μmID) and separated using a preset 30 samples / day gradient with the Evosep One system. MS data were acquired using Xcalibur software. The top 10 most abundant precursor ions were dynamically selected from investigative scans using HCD fragmentation, employing a data-dependent method. Investigational scans were acquired at m / z 200 with a resolution of 60,000 in the mass range of 400–1000 Th. The maximum ion implantation times for the investigational scan and MS / MS scan were 50 ms and 100 ms, respectively, and the AGC target values ​​were set to 3E6 and 1E5, respectively. The separation window was set to 1.5 Th, and ions were fragmented with a normalized collision energy of 27. Charge states of unassigned precursor ions, monovalent ions, and ions with charge states greater than 8 were excluded. Peptide matching was preferred, and dynamic exclusion was set to 40 seconds.

[0080] Examples of bioinformatics analysis Mass spectra were analyzed using MaxQuant software version 1.6.6.0. The maximum allowable mass deviation was set to 4.5 ppm for monoisotopic precursor ions and 0.5 Da for MS / MS peaks. Enzyme specificity was set to trypsin / P, allowing for a maximum of two cleavage errors. Carbamide methylcysteine ​​was set as the fixed modification, and N-terminal acetylation and methionine oxidation were set as variable modifications. Spectra were searched against the human Uniprot sequence database, concatenated with the reverse versions of all sequences, combined with common contaminants. Protein identification required at least one unique peptide or laser peptide per protein group. MaxQuant quantification was performed using the label-free quantification (LFQ) algorithm with fast LFQ and a minimum ratio count of 1. The false-positive rate was set to 1% at both the peptide and protein levels. Run-to-run agreement was selected using a 20-minute alignment time window and a 0.7-minute agreement time window. Contaminants, proteins identified solely by reverse sequence identification, and site identification were excluded from further data analysis. Missing values ​​were substituted with the minimum LFQ intensity of the sample.

[0081] IPA Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA) software (Qiagen; Hilden, Germany; www.qiagen.com / ingenuity). See Kramer A, Green J, Pollard Jr J, Tugendreich S. Causal analysis approaches in ingenuity pathway analysis. Bioinformatics. 2014;30(4):523-30. UPPs were identified according to their UniProt specific annotations and included in the overrepresentation analysis if they were associated with the results at a nominal p-value threshold of 0.01. This analysis calculates p-values ​​(Fisher's exact test) to quantify overlap and z-scores to quantify the likelihood and direction (upward or downward control) between proteomics patterns and known canonical pathways.

[0082] statistical analysis Participant characteristics were assessed using the mean and standard deviation (SD) for normally distributed variables, and the median and interquartile range (IQR) for non-normally distributed variables. Clinical features were compared between subjects for whom urinary proteomics data was available and those for whom it was not. Unpaired Student's t-tests were used for normally distributed continuous variables, Kruskal-Wallis tests for non-normally distributed continuous variables, and chi-squared tests or Fisher's exact test for categorical variables, as needed.

[0083] The primary outcome of this analysis was a composite of death or hospitalization for heart failure (DHFA), as previously defined and used. See Chirinos JA, Zhao L, Jia Y, Frej C, Adamo L, Mann D, et al. Reduced Apolipoprotein M and Adverse Outcomes Across the Spectrum of Human Heart Failure. Circulation. 2020;141(18):1463-76. Cox regression was used to assess the relationship between urinary biomarker levels and the risk of DHFA. Three different analyses were performed to assess the impact of adjustments for different clinical factors. These included 1) an unadjusted model, 2) a model adjusted for urinary creatinine levels, and 3) a model adjusted for the Meta-Analysis Global Group (MAGGIC) risk score in chronic HF, incorporating urinary creatinine levels and multiple demographic, clinical, and laboratory parameters. See Pocock SJ, Ariti CA, McMurray JJ, Maggioni A, Kober L, Squire IB, et al. Predicting survival in heart failure: a risk score based on 39,372 patients from 30 studies. Eur Heart J. 2013;34(19):1404-13. Statistical significance was defined as a two-sided p-value < 0.05. Alpha levels for multiple comparisons were corrected based on the principal components underlying the variability of all measured UPPs. See Tromp J, Khan MA, Klip IT, Meyer S, de Boer RA, Jaarsma T, et al. Biomarker Profiles in Heart Failure Patients With Preserved and Reduced Ejection Fraction. J Am Heart Assoc. 2017;6(4). All probability values ​​shown are two-sided. Analysis was performed using MATLAB Statistics and the Machine Learning Toolbox R2022a.

[0084] result TOPCAT Group TOPCAT urinary proteomics was performed using a low-input, high-throughput workflow. Small 0.35 mL urine samples were collected from patients with protein concentrations between <50 ug / ml and >5000 ug / ml. One-plate sample preparation was performed using protein precipitation and enzymatic digestion. Peptide cleanup was performed using the iST-BCT kit, and total peptide measurement was performed by tryptophan fluorescence assay. Final measurements were performed using nanoLCBMS with low input (400 ng peptide / injection) and high throughput (1.5 days per plate). Unbiased relative protein quantification of over 2300 proteins was achieved across the entire cohort.

[0085] Urinary protein data from a total of 426 participants (12.4%) from TOPCAT were included in this analysis. The TOPCAT trial was a multicenter (233 centers, 6 countries), randomized, double-blind trial involving 3,445 patients with symptomatic heart failure (HF) and a left ventricular ejection fraction (LVEF) of 45% or higher. Patients received either placebo (n=1,723) or spironolactone (n=1,722, 15-45 mg / day).

[0086] Table 1 shows a comparison between subjects with and without available urinary protein data. There were no statistically significant differences in age, race, glomerular filtration rate, or country between subjects with and without protein data. In general, individuals with urinary proteomics data were more likely to be male and had a slightly higher body mass index (BMI). Those with urinary proteomics data also tended to have a slightly higher prevalence of atrial fibrillation, a history of myocardial infarction, and hypertension, and were somewhat more likely to use angiotensin-converting enzyme inhibitors / angiotensin II receptor blockers and statins. Although the quantitative differences were small (130 mmHg vs. 128 mmHg between participants with and without urine samples), these participants showed a significant decrease in systolic blood pressure.

[0087] [Table 1]

[0088] Association between UPP levels and DHFA incidence: Unadjusted analysis In the unadjusted analysis, the inventors found that 40 urinary proteins / peptides were significantly associated with DHFA after alpha error correction. Figure 1A shows a volcano plot illustrating the relationship between UPP and DHFA risk. Table 2 lists the UPPs that were significantly associated with DHFA, along with their standardized HR and 95% CI. Table 3 lists the names, functions, and categories of these proteins in complete detail.

[0089] In the unadjusted analysis, after alpha error correction for multiple comparisons, the inventors found that 40 urinary proteins were significantly associated with DHFA, and 21 of these were also significantly associated in the adjusted analysis (Figure 1B). Several of the identified proteins are involved in fibrosis, inflammation, renal sodium processing, and metabolism.

[0090] [Table 2-1] [Table 2-2] [Table 2-3]

[0091] Table 2 shows the statistical association between urinary protein levels and results in either the unadjusted analysis (left) or the analysis adjusted for urinary creatinine levels and MAGGIC risk scores (right).

[0092] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0093] Of the 40 UPPs associated with DHFA, eight showed a positive association, while 32 showed an inverse association with DHFA risk. Elevated levels of the following eight proteins were associated with an increased risk of DHFA: methyl CpG-binding domain protein 1 (MBD1, K7EPZ6), immunoglobulin heavy chain constant alpha 2 (IGHA2, A0A075B6N7), albumin (P02768), immunoglobulin lambda variable 3-25 (IGLV3-25, P01717), melanoma-associated antigen 4 (MAGEA4, P43358), kinesin-like proteins (KIF3A, J3KPF9), APCS (P02743), and haptoglobin (P00738). For the following 32 proteins, elevated urinary levels were associated with a reduced risk of DHFA: COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

[0094] The top prognostic uppants are deoxyribonuclease 1 (DNASE1) (HR=0.584, CI=0.46-0.69, P=2.09E-08), alpha-amylase 2A (AMY2A) (HR=0.588, CI=0.460.69, P=2.34E-08), angiopoietin-like protein 2 (ANGPTL2) (HR=0.566, CI=0.48-0.71, P=7.54E-08), and epidermal growth factor ( EGF) (HR=0.576, CI=0.47-0.71, P=1.33E-07), Zymogen granule protein 16B (ZG16B) (HR=0.594, CI=0.49-0.73, P=0.0001), Aldehyde dehydrogenase 1 family member A1 (ALDH1A1) (HR=0.599, CI=0.49-0.73, P=0.0001), Inducible T cell costimulatory ligand (ICOSLG) (HR (HR=0.611, CI=0.5-0.74, P=0.0002), Plasminogen-activated urokinase (PLAU) (HR=0.595, CI=0.49-0.73, P=0.0002), Heat shock protein 60 (HSP60) (HR=0.602, CI=0.49-0.74, P=0.0002), Alpha-amylase 2B (AMY2B) (HR=0.594, CI=0.48-0.73, P=0.000) 2) These were beta-1,4-galactosyltransferase 1 (B4GALT1) (HR=0.604, CI=0.49-0.74, P=0.0003), solute carrier family 2 member 5 (SLC2A5) (HR=0.612, CI=0.5-0.75, P=0.0004), and collagen alpha 1 (XV) chain (COL15A1) (HR=0.637, CI=0.53-0.77, P=0.0014).

[0095] UPPs that show a positive association with DHFA risk include immunoglobulin lambda variable 3-25 (IGLV3-25) (HR=1.576, CI=1.28-1.94, P=0.0065), serum amyloid P component (APCS) (HR=1.547, CI=1.27-1.88, P=0.0044), and methyl CpG-binding domain protein 1 (MBD1) (HR=1.5 The study included 2 (CI=1.23-1.88, P=0.0317), albumin (HR=1.495, CI=1.23-1.82, P=0.0196), haptoglobin (HR=1.495, CI=1.22-1.83, P=0.0363), and immunoglobulin heavy chain constant alpha-2 (IGHA2) (HR=1.458, CI=1.2-1.77, P=0.045). All other UPPs showed a negative association with DHFA.

[0096] Association between urinary biomarker levels and DHFA incidence: Adjusted analysis In a model adjusted for urinary creatinine, the inventors found that 39 proteins were significantly associated with the incidence of DHFA. All but one (glutathione S-transferase Mu 3, P21266) were also significantly associated with the results of the unadjusted analysis. Glutathione S-transferase Mu 3 was downregulated with Std. beta-0.37 and HR 0.6.

[0097] In a model adjusted for urinary creatinine and MAGGIC scores, the inventors found that 21 UPPs were significantly associated with DHFA risk, and all of these were significantly associated with this result in the unadjusted analysis. Peptides / proteins correlated with DHFA (upregulated proteins) include MBD1 and IGHA2. Peptides / proteins inversely correlated with DHFA include ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868. Proteins ANGPTL2, AMY2A, and SLC2A5 were identified as the target subset for measuring downregulated proteins.

[0098] In these adjusted models, the top UPPs associated with DHFA risk included: angiopoietin-like protein 2 (ANGPTL2) (HR=0.5731, CI=0.47-0.7, P=3.13E-05), alpha-amylase 2A (AMY2A) (HR=0.5496, CI=0.44-0.69, P=0.0001), deoxyribonuclease 1 (DNASE1) (HR=0.5704, CI=0.46-0.71, P=0.0002), aldehyde dehydrogenase 1 family member A1 (ALDH1A1) (HR=0.5572, CI=0.47-0.72, P=0.0003), and alpha-amylase 2B (AMY2B) (HR=0.0 0.5841, CI=0.44-0.7, P=0.0003), Plasminogen-activated urokinase (PLAU) (HR=0.5898, CI=0.47-0.74, P=0.0016), Heat shock protein 60 (HSP60) (HR=0.6188, HR=0.5-0.76, P=0.0017), Zymogen granule protein 16 B(ZG16B) (HR=0.6137, CI=0.5-0.76, P=0.0018), inducible T cell costimulatory ligand (ICOSLG) (HR=0.6322, CI=0.51-0.78, P=0.0067), and phosphoglycerate kinase 1 (PGKA) (HR=0.6507, CI=0.53-0.8, P=0.0101).

[0099] IPA Path Analysis Overexpression analysis of the IPA pathway based on UPP associated with DHFA risk identified five standard signaling pathways associated with this result. Four of these were fibrosis-related pathways (hepatic fibrosis / hepatic stellate cell activation, wound healing and idiopathic pulmonary fibrosis, and GP6 signaling), while the remaining pathway (NAD signaling) was related to cellular metabolism.

[0100] Consideration The inventors conducted a proteomic analysis of urinary biomarkers associated with DHFA risk in HFpEF. Based on these results, they identified 40 urinary proteins / peptides associated with fibrosis, metabolism, and inflammation. After adjusting for MAGGIC risk scores and urinary creatinine levels, they identified 21 proteins associated with DHFA risk.

[0101] The inventors have identified several UPPs that have been shown to show an inverse association with the risk of DHFA in HFpEF. Interestingly, high serum levels of some of these proteins were associated with poor outcomes in other populations. For example, in our studies, elevated urinary ANGPTL2 levels were associated with a reduced risk of DHFA, while prior literature has linked ANGPTL2 to its role in cardiovascular disease and HF due to its role in chronic inflammation. Elevated serum ANGPTL2 levels in pathologically stressed hearts accelerate cardiac dysfunction, but inhibiting ANGPTL2 may delay disease progression. The inventors also found that urinary urokinase-type plasminogen activator (PLAU or suPAR) levels showed an inverse association with the risk of DHFA. In previous studies, plasma levels of suPAR were associated with cardiovascular death or MI in patients with coronary artery disease (CAD) and congestive HF (primarily consisting of HFrEF patients).

[0102] Notably, plasma and urine levels of protein generally show a low correlation with each other. While urine is primarily produced as a result of plasma filtration by the kidneys, the origin of proteins and peptides present in urine is not as clear.

[0103] Elevated levels of fibrosis peptides in urine are associated with a reduced risk of DHFA. The inventors discovered that elevated urinary levels of two collagen-derived proteins, COL15A1 (collagen alpha-1(XV) chain) and COL6A1 (collagen alpha-1(VI) chain), were associated with a reduced risk of DHFA. COL6A1 is a type VI collagen chain and a major structural component of microfibrils. Interestingly, plasma levels of endotrophin, a peptide derived from collagen VI alpha-3 chain, have been reported to be strongly and positively associated with the risk of adverse outcomes in HFpEF. COL15A1 is part of type XV collagen and is widely expressed, but is also often localized to the basement membrane. Collagen turnover and tissue impairment in cardiomyopathy are well established. They are also specifically linked as part of the pathophysiology of HFpEF. In the inventors' studies on urinary proteomics, elevated levels of urinary biomarkers associated with collagen metabolism, including COL6A1 and COL15A1, were generally enriched in patients with both HFpEF and HFrEF. However, the association with those outcomes was not evaluated in this study. See He T, et al. Urinary peptides in heart failure: a link to molecular pathophysiology. European Journal of Heart Failure. 2021;23(11):1875-87.

[0104] Our findings regarding the inverse association between COL6A1 and COL15A1 and the risk of DHFA are counterintuitive, given the association between plasma biomarkers of histofibrosis and adverse outcomes of HFpEF. It should be noted that urinary protein / peptide levels have multiple determinants and do not directly represent plasma levels. In addition to glomerular filtration, proteins can be secreted in the ureters by tubular and epithelial cells. For example, collagen secretion by tubular and epithelial cells has been reported to be affected by albumin exposure. Furthermore, there is variability in the reabsorption of filtered proteins by tubular cells, and there may be individual differences in the degradation of filtered or secreted proteins, ultimately affecting the measured level of protein in urine. Finally, elevated urinary levels may represent increased clearance of plasma collagen-derived peptides.

[0105] Metabolic pathways involved in the outcome The inventors have identified several UPPs related to metabolic processes, including carbohydrate metabolism (AMY2A: pancreatic alpha-amylase, AMY2B: alpha-amylase 2B, MAN1A1: mannosyl oligosaccharide 1,2-alpha-mannosidase IA) and lipid metabolism (ALDH1A1: aldehyde dehydrogenase 1A1, B4GALT1: beta-1,4-galactosyltransferase 1).

[0106] The inventors discovered that increased urinary protein levels of pancreatic alpha-amylase (AMY2A and AMY2B) are associated with a reduced risk of DHFA. While the literature on the association between AMY2A and HF is limited, one study found that plasma AMY2A levels were lower in HF patients than in healthy controls. However, another study reported that plasma amylase levels were elevated in patients with severe HF but not in patients with mild HF. Notably, urinary amylase assays are widely used for their role in the diagnosis of acute pancreatitis.

[0107] Other new relevances The inventors have discovered prostasin, which shows an inverse association with the risk of DHFA. Prostasin is an epithelial sodium channel stimulant. Reduced prostasin expression is associated with poor outcomes in colorectal cancer and oral squamous cell carcinoma. However, while recent studies have found plasma prostasin levels to be positively associated with diabetes risk and cancer mortality, another study has found serum prostasin to be inversely associated with physical activity in population-based cohorts. It is unclear whether the association between decreased urinary prostasin levels and increased DHFA risk observed in the inventors' studies is related to the physical activity of these patients. To the best of the inventors' knowledge, prostasin is not associated with HFpEF.

[0108] Limitations of the test The inventors' study should be interpreted in relation to its advantages and limitations. The advantages of the inventors' study include a well-characterized cohort, a relatively long follow-up period, prospectively adjudicated results based on rigorous criteria and methodology, and the unbiased nature of UPP measurement. The inventors' study also has limitations. Urine samples were not available from all TOPCAT participants. Furthermore, the inclusion of some participants with undiagnosed cardiac amyloidosis and / or renal amyloidosis could not be excluded, potentially leading to confusion with urinary proteome. Given the limited availability of prospective follow-up and HFpEF cohorts with available urine samples, this study did not include an external validation cohort. Finally, the origin of UPP measured in the inventors' study (contributions from circulating blood, urinary tract secretion, differential catabolism, etc.) could not be evaluated.

[0109] conclusion Our studies have reported a relationship between UPP and DHFA risk in HFpEF. We have identified several novel associations between UPP and adverse outcomes in this patient population. High levels of urinary protein involved in fibrosis, metabolism, and inflammation are associated with a lower risk of composite DHFA outcomes in HFpEF. Many of these novel associations are independent of clinical risk scores and may be useful for risk stratification in HFpEF.

Claims

1. A method for determining the risk of death or hospitalization for heart failure (DHFA) in a subject, comprising: measuring or having measured one or more levels of urinary protein in the urine of the subject; analyzing or having analyzed the one or more levels of urinary protein; and determining the risk of DHFA in the subject based on the analysis of the one or more levels of urinary protein.

2. The method according to claim 1, wherein the subject is suffering from and / or has been diagnosed with heart failure with preserved ejection fraction (HFpEF).

3. The levels of one or more of the aforementioned urinary proteins include MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, haptoglobin, COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, The method according to claim 1, comprising levels of one or more proteins selected from the group consisting of HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, Prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

4. The method according to claim 3, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868.

5. The method according to claim 1, wherein the analysis includes determining whether one or more of the urinary protein levels are elevated.

6. The method according to claim 5, comprising determining that the risk of DHFA in the subject is increased if one or more of the urinary protein levels are elevated.

7. The method according to claim 6, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, and haptoglobin.

8. The method according to claim 7, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1 and IGHA2.

9. The method according to claim 5, comprising determining that the risk of DHFA in the subject is reduced if one or more of the urinary protein levels are elevated.

10. The method according to claim 9, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

11. The method according to claim 10, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868.

12. The method according to claim 11, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, and SLC2A5.

13. The method according to any prior claim, further comprising obtaining a urine sample from the subject, or having obtained one or more urine protein levels from the sample.

14. The method according to any of the prior claims, wherein the analysis of the one or more urinary protein levels comprises comparing the one or more urinary protein levels to a threshold.

15. The method according to any prior claim, wherein the HFpEF is a highly contractile HFpEF.

16. The method according to any prior claim, wherein the HFpEF is a low-contractile HFpEF.

17. The method according to any prior claim, further comprising treating the subject with a certain therapy based on the DHFA risk of the subject.

18. The method according to claim 17, wherein the therapy is HFpEF therapy.

19. The method according to claim 18, wherein the therapy is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), SGLT2 inhibitors, diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine, or any combination thereof.

20. The method according to any of the prior claims, wherein the subject has symptomatic heart failure and a left ventricular ejection fraction (LVEF) of 45% or more.

21. The method according to claim 13, wherein the urine sample is frozen after acquisition and before measurement of the urine protein level.

22. The method according to any of the prior claims, wherein the level of one or more urinary proteins in the urine of the subject is measured using liquid chromatography-mass spectrometry (LC-MS).

23. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more collagen-derived proteins.

24. The method according to claim 23, wherein the one or more collagen-derived proteins include COL15A1 and / or COL6A1.

25. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more pancreatic alpha-amylases.

26. The method according to claim 25, wherein the one or more pancreatic alpha-amylases include AMY2A.

27. The method according to claim 25, wherein the one or more pancreatic alpha-amylases include AMY2B.

28. The method according to any prior claim, wherein the one or more urinary protein levels include the levels of one or more urinary proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, and AMY2B.

29. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ANGPTL2.

30. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of DNASE1.

31. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ALDH1A1.

32. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of SLC2A5.

33. The method according to any prior claim, wherein the one or more urinary protein levels include a prostacin level.

34. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of DNASE1, AMY2A, ANGPTL2, EGF, ZG16B, ALDH1A1, ICOSLG, PLAU, HSP60, AMY2B, B4GALT1, SLC2A5, and COL15A1.

35. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, AMY2B, PLAU, HSP60, ZG16B, ICOSLG, and PGKA.

36. A method for treating heart failure with preserved ejection fraction (HFpEF) in patients requiring treatment, the following: Measuring or having measured the level of one or more urinary proteins in the urine of the subject; Analyzing, or having analyzed, one or more of the aforementioned urinary protein levels; Determining, or having determined, the risk of death or hospitalization for heart failure (DHFA) in the subject based on the analysis of one or more urinary protein levels; and Treating the subject with HFpEF therapy, Methods that include...

37. The levels of one or more of the aforementioned urinary proteins include MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, haptoglobin, COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, H The method according to claim 36, comprising a level of one or more proteins selected from the group consisting of SP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, Prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

38. The method according to claim 37, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868.

39. The method according to claim 36, wherein the analysis includes determining whether one or more urinary protein levels are elevated.

40. The method according to claim 39, comprising determining that the risk of DHFA in the subject is increased if one or more of the urinary protein levels are elevated.

41. The method according to claim 40, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, and haptoglobin.

42. The method according to claim 41, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1 and IGHA2.

43. The method according to claim 39, comprising determining that the risk of DHFA in the subject is reduced if one or more of the urinary protein levels are elevated.

44. The method according to claim 43, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

45. The method according to claim 44, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868.

46. The method according to claim 45, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, and SLC2A5.

47. The method according to any prior claim, further comprising obtaining a urine sample from the subject, or having obtained one or more urine protein levels from the sample.

48. The method according to any of the prior claims, wherein the analysis of the one or more urinary protein levels comprises comparing the one or more urinary protein levels to a threshold.

49. The method according to any prior claim, wherein the HFpEF is a highly contractile HFpEF.

50. The method according to any prior claim, wherein the HFpEF is a low-contractile HFpEF.

51. The method according to any prior claim, wherein the HFpEF therapy is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), SGLT2 inhibitors, diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine, or any combination thereof.

52. The following treatments: Measuring, or having measured, one or more post-treatment urinary protein levels in the urine of the subject; Analyzing, or having analyzed, the urinary protein levels after one or more of the aforementioned treatments; and Based on the analysis of one or more urinary protein levels, determine or have determined the post-treatment risk of DHFA in the subject. The method according to any prior claim, further comprising:

53. The method according to any of the prior claims, wherein the subject has symptomatic heart failure and a left ventricular ejection fraction (LVEF) of 45% or more.

54. The method according to claim 47, wherein the urine sample is frozen after acquisition and before measurement of the urine protein level.

55. The method according to any of the prior claims, wherein the level of one or more urinary proteins in the urine of the subject is measured using liquid chromatography-mass spectrometry (LC-MS).

56. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more collagen-derived proteins.

57. The method according to claim 56, wherein the one or more collagen-derived proteins include COL15A1 and / or COL6A1.

58. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more pancreatic alpha-amylases.

59. The method according to claim 58, wherein the one or more pancreatic alpha-amylases include AMY2A.

60. The method according to claim 58, wherein the one or more pancreatic alpha-amylases include AMY2B.

61. The method according to any prior claim, wherein the one or more urinary protein levels include the levels of one or more urinary proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, and AMY2B.

62. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ANGPTL2.

63. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of DNASE1.

64. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ALDH1A1.

65. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of SLC2A5.

66. The method according to any prior claim, wherein the one or more urinary protein levels include a prostacin level.

67. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of DNASE1, AMY2A, ANGPTL2, EGF, ZG16B, ALDH1A1, ICOSLG, PLAU, HSP60, AMY2B, B4GALT1, SLC2A5, and COL15A1.

68. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, AMY2B, PLAU, HSP60, ZG16B, ICOSLG, and PGKA.

69. The method according to any of the prior claims, wherein if the subject is determined to be at risk of DHFA, a first HFpEF therapy is administered, and if the subject is determined not to be at risk of DHFA, a second HFpEF therapy is administered.

70. The method according to any of the prior claims, wherein if the subject is determined to be at high risk of DHFA, a first HFpEF therapy is administered, and if the subject is determined to be at low risk of DHFA, a second HFpEF therapy is administered.

71. The method according to claim 69 or 70, wherein the first HFpEF therapy and the second HFpEF therapy each comprise the administration of the same therapeutic agent, and the first HFpEF therapy comprises the administration of a higher dose of the therapeutic agent compared to the second HFpEF therapy.

72. The method according to claim 69 or 70, wherein the first HFpEF therapy comprises the administration of a therapeutic agent different from the second HFpEF therapy.

73. The method according to any one of claims 69 to 72, wherein the first HFpEF therapy is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine, or pharmaceutically acceptable salts thereof, or any combination thereof.

74. The method according to any one of claims 69 to 72, wherein the first HFpEF therapy is an SGLT2 inhibitor.

75. The method according to any one of claims 69 to 72, wherein the first HFpEF therapy is afficamten or a pharmaceutically acceptable salt thereof.

76. The method according to any one of claims 69 to 75, wherein the second HFpEF therapy is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine, or any combination thereof.

77. A method for evaluating the severity of HFpEF in a subject, the following: Measuring or having measured the level of one or more urinary proteins in the urine of the subject; Analyzing, or having analyzed, one or more of the aforementioned urinary protein levels; and Based on the analysis of one or more urinary protein levels, the severity of HFpEF in the subject is determined. Methods that include...

78. The method according to claim 77, wherein the severity of the HFpEF is characterized by the risk of death or hospitalization for heart failure (DHFA) in the subject.

79. A method for monitoring the severity of HFpEF in subjects who have HFpEF or have been diagnosed with HFpEF, the following: Measuring or having measured the level of one or more urinary proteins in the urine of the subject; Analyzing, or having analyzed, one or more of the aforementioned urinary protein levels; and Based on the analysis of one or more urinary protein levels, the severity of HFpEF in the subject is determined. Methods that include...

80. The method according to claim 79, wherein the severity of the HFpEF is characterized by the risk of death or hospitalization for heart failure (DHFA) in the subject.

81. A method for identifying treatment options for individuals at risk of death or hospitalization for heart failure (DHFA), which includes the following: (a) Obtaining or having obtained a urine sample from the subject; (b) Measuring or having measured one or more levels of urinary protein in the sample; (c) Analyzing the level of urinary protein in the sample, or having done so; (d) Selecting at least one urinary protein based on the analysis; and (e) Selecting a drug or combination drug (where the drug or combination drug has a functional effect on the at least one urinary protein selected in (d)), Methods that include...

82. (f) The method according to claim 81, further comprising administering the drug or combination drug to the subject.

83. (g) The method according to claim 82, further comprising measuring at least one urinary protein selected in (d) after administration of the drug or combination drug.

84. The method according to any prior claim, wherein the drug or combination drug is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, ivabradine, SGLT2 inhibitors, and afficamten, or pharmaceutically acceptable salts thereof, or any combination thereof.

85. A method for treating heart failure with preserved ejection fraction (HFpEF) in patients requiring treatment, the following: Measuring or having measured the level of one or more urinary proteins in the urine of the subject; Analyzing, or having analyzed, one or more of the aforementioned urinary protein levels; Determining, or having determined, the severity of HFpEF in the subject based on the analysis of one or more urinary protein levels; and The subject to be treated with HFpEF therapy. Methods that include...

86. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more collagen-derived proteins.

87. The method according to any of the prior claims, wherein the one or more collagen-derived proteins include COL15A1 and / or COL6A1.

88. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more pancreatic alpha-amylases.

89. The method according to any of the prior claims, wherein the one or more pancreatic alpha-amylases include AMY2A.

90. The method according to any of the prior claims, wherein the one or more pancreatic alpha-amylases include AMY2B.

91. The method according to any prior claim, wherein the one or more urinary protein levels include the levels of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, and AMY2B.

92. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ANGPTL2.

93. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of DNASE1.

94. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ALDH1A1.

95. The method according to any of the prior claims, wherein the one or more urinary protein levels include the levels of MBD1 and / or IGHA2.

96. The method according to any prior claim, wherein the one or more urinary protein levels include levels of ANGPTL2, AMY2A, SLC2A5, or a combination thereof.

97. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of SLC2A5.

98. The method according to any prior claim, wherein the one or more urinary protein levels include a prostacin level.

99. The levels of one or more of the aforementioned urinary proteins include MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, haptoglobin, COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, and HSP. The method according to any prior claim, comprising a level of one or more proteins selected from the group consisting of 60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, Prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

100. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, and haptoglobin.

101. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

102. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of DNASE1, AMY2A, ANGPTL2, EGF, ZG16B, ALDH1A1, ICOSLG, PLAU, HSP60, AMY2B, B4GALT1, SLC2A5, and COL15A1.

103. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868.

104. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, AMY2B, PLAU, HSP60, ZG16B, ICOSLG, and PGKA.

105. The method according to any prior claim, wherein the HFpEF therapy is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, ivabradine, SGLT2 inhibitors, and afficamten, or pharmaceutically acceptable salts thereof, or any combination thereof.

106. A method for determining the risk of death or hospitalization for heart failure (DHFA) in a subject, comprising determining the risk of DHFA in the subject based on one or more urinary protein levels.

107. The method according to any prior claim, further comprising measuring, or having measured, one or more levels of urinary protein in the urine of the subject.

108. The method according to any prior claim, wherein the subject is suffering from and / or has been diagnosed with heart failure with preserved ejection fraction (HFpEF).

109. The method according to any of the prior claims, wherein the subject is suffering from symptomatic heart failure and / or has been diagnosed with symptomatic heart failure.

110. The method according to any of the prior claims, wherein the subject is suffering from and / or has been diagnosed with NYHA class II, III, or IV heart failure.

111. The method according to any of the prior claims, wherein determining the risk of DHFA includes determining whether one or more urinary protein levels are above or below a threshold.

112. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more collagen-derived proteins.

113. The method according to any of the prior claims, wherein the one or more collagen-derived proteins include COL15A1 and / or COL6A1.

114. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more pancreatic alpha-amylases.

115. The method according to any of the prior claims, wherein the one or more pancreatic alpha-amylases include AMY2A.

116. The method according to any of the prior claims, wherein the one or more pancreatic alpha-amylases include AMY2B.

117. The method according to any prior claim, wherein the one or more urinary protein levels include the levels of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, and AMY2B.

118. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ANGPTL2.

119. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of DNASE1.

120. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ALDH1A1.

121. The method according to any of the prior claims, wherein the one or more urinary protein levels include the levels of MBD1 and / or IGHA2.

122. The method according to any prior claim, wherein the one or more urinary protein levels include levels of ANGPTL2, AMY2A, SLC2A5, or a combination thereof.

123. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of SLC2A5.

124. The method according to any prior claim, wherein the one or more urinary protein levels include a prostacin level.

125. The levels of one or more of the aforementioned urinary proteins include MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, haptoglobin, COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, and HSP. The method according to any prior claim, comprising a level of one or more proteins selected from the group consisting of 60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, Prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

126. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, and haptoglobin.

127. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

128. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of DNASE1, AMY2A, ANGPTL2, EGF, ZG16B, ALDH1A1, ICOSLG, PLAU, HSP60, AMY2B, B4GALT1, SLC2A5, and COL15A1.

129. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868.

130. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, AMY2B, PLAU, HSP60, ZG16B, ICOSLG, and PGKA.

131. A method for treating heart failure with preserved ejection fraction (HFpEF) in patients requiring treatment, the following: Determining one or more urinary protein levels in the aforementioned subjects; and If one or more of the aforementioned urinary protein levels exceed the threshold, the subject shall be administered the first HFpEF therapy, and If one or more of the aforementioned urinary protein levels are below the threshold, the subject shall be administered a second HFpEF therapy. Methods that include...

132. A method for treating heart failure with preserved ejection fraction (HFpEF) in a patient requiring treatment, wherein the patient is administered a first HFpEF therapy, and the following applies: Determining one or more urinary protein levels in the aforementioned subjects; and If one or more of the aforementioned urinary protein levels exceed the threshold, the subject shall be administered a second HFpEF therapy. Methods that include...

133. A method for treating heart failure with preserved ejection fraction (HFpEF) in a patient requiring treatment, wherein the patient is administered a first HFpEF therapy, and the following applies: Determining one or more urinary protein levels in the aforementioned subjects; and If one or more of the aforementioned urinary protein levels are below the threshold, the subject shall be administered a second HFpEF therapy. Methods that include...

134. The method according to any prior claim, wherein the second HFpEF therapy comprises a drug different from the first therapy.

135. The method according to any of the prior claims, wherein the first HFpEF therapy comprises a first drug in a first dosage, and the second therapy comprises the first drug in a second dosage.

136. The method according to any prior claim, wherein the first HFpEF therapy is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine, or pharmaceutically acceptable salts thereof, or any combination thereof.

137. The method according to any of the prior claims, wherein the first HFpEF therapy is an SGLT2 inhibitor.

138. The method according to any prior claim, wherein the first HFpEF therapy is afficamten or a pharmaceutically acceptable salt thereof.

139. The method according to any prior claim, wherein the second HFpEF therapy is selected from the group consisting of beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), angiotensin receptor neprilysin (ARN) inhibitors, mineralocorticoid receptor antagonists (MRAs), diuretics, GLP-1 agonists, calcium sensitizers, soluble guanylate cyclase stimulants, and ivabradine, or pharmaceutically acceptable salts thereof, or any combination thereof.

140. The method according to any prior claim, wherein the subject is suffering from and / or has been diagnosed with heart failure with preserved ejection fraction (HFpEF).

141. The method according to any of the prior claims, wherein the subject is suffering from symptomatic heart failure and / or has been diagnosed with symptomatic heart failure.

142. The method according to any of the prior claims, wherein the subject is suffering from and / or has been diagnosed with NYHA class II, III, or IV heart failure.

143. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more collagen-derived proteins.

144. The method according to any of the prior claims, wherein the one or more collagen-derived proteins include COL15A1 and / or COL6A1.

145. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of one or more pancreatic alpha-amylases.

146. The method according to any of the prior claims, wherein the one or more pancreatic alpha-amylases include AMY2A.

147. The method according to any of the prior claims, wherein the one or more pancreatic alpha-amylases include AMY2B.

148. The method according to any prior claim, wherein the one or more urinary protein levels include the levels of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, and AMY2B.

149. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ANGPTL2.

150. The method according to any of the prior claims, wherein the one or more urinary protein levels include the level of DNASE1.

151. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of ALDH1A1.

152. The method according to any of the prior claims, wherein the one or more urinary protein levels include the levels of MBD1 and / or IGHA2.

153. The method according to any prior claim, wherein the one or more urinary protein levels include levels of ANGPTL2, AMY2A, SLC2A5, or a combination thereof.

154. The method according to any of the prior claims, wherein one or more urinary protein levels include the level of SLC2A5.

155. The method according to any prior claim, wherein the one or more urinary protein levels include a prostacin level.

156. The levels of one or more of the aforementioned urinary proteins include MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, haptoglobin, COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, and HSP. The method according to any prior claim, comprising a level of one or more proteins selected from the group consisting of 60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, Prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

157. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, albumin, IGLV3-25, MAGEA4, KIF3A, APCS, and haptoglobin.

158. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of COL15A1, COL6A1, ZG16B, ICOSLG, XPNPEP2, attractin, ALDH1A1, PGKA, PLAU, EGF, AMY2A, ATP1B1, SERPINA5, MELTF, HSP60, B4GALT1, CPE, AMY2B, SLC2A5, PROZ, DNASE1, ATP5F1A, MAN1A1, IGFALS, SLC12A3, prostasin, QPCT, HMCN1, C11orf54, LRRC19, ANGPTL2, and X6R868.

159. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of DNASE1, AMY2A, ANGPTL2, EGF, ZG16B, ALDH1A1, ICOSLG, PLAU, HSP60, AMY2B, B4GALT1, SLC2A5, and COL15A1.

160. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of MBD1, IGHA2, ANGPTL2, AMY2A, DNASE1, AMY2B, ALDH1A1, PLAU, HSP60, ZG16B, ICOSLG, EGF, PGKA, C11orf54, ATP5F1A, SLC2A5, prostasin, ATP1B1, COL6A1, B4GALT1, and X6R868.

161. The method according to any prior claim, wherein the one or more urinary protein levels include the level of one or more proteins selected from the group consisting of ANGPTL2, AMY2A, DNASE1, ALDH1A1, AMY2B, PLAU, HSP60, ZG16B, ICOSLG, and PGKA.

162. The method according to any of the prior claims, wherein the subject is determined to be at risk of HFpEF before the onset of HFpEF.

163. The method according to any of the prior claims, wherein the subject is suffering from and / or has been diagnosed with hypertrophic cardiomyopathy (HCM) and is considered to be at risk of HFpEF.

164. The method according to any of the prior claims, wherein the subject has NYHA class I heart failure and / or has been diagnosed with NYHA class I heart failure and is considered to be at risk of HFpEF.

165. The method according to any prior claim, wherein the subject is an HFpEF patient who is resistant to drug therapy based on guidelines.

166. The method according to any prior claim, wherein the subject is an HFpEF patient who is intolerant to drug therapy based on guidelines.

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