Measurement of urine thromboxane metabolites to predict survival

Abstract

The present disclosure provides a method of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample, including determining the amount of thromboxane B2 metabolites (TXB2-M) in the urine sample, adjusting or normalizing the amount of thromboxane B2 metabolites (TXB2-M) to creatinine (e.g., urine creatinine or creatinine level) in the urine sample (e.g., picogram (pg) of thromboxane B2 metabolites (TXB2-M) / milligram (mg) of creatinine), and adjusting or normalizing the creatinine (e.g., pg / mg) in the urine sample to renal function. The present disclosure further comprises methods of examining or determining mortality risk or hear failure risk in a subject, as well as methods of treating subjects (i) at increased risk for heart failure and / or (ii) at increased mortality risk of cardiovascular disease, heart failure, and / or stroke.

Description

MEASUREMENT OF URINE THROMBOXANE METABOLITES TO PREDICTSURVIVALCROSS-REFENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 559,030, filed 28 February 2024, which is incorporate by reference herein in its entirety for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under contracts NO1-HC- 25195, HHSN268201500001I and 75N92019D00031 from the National Heart, Lung, and Blood Institute. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present disclosure provides methods of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample, methods of examining or determining mortality risk in a subject, and methods of treating a subject at increased mortality risk.BACKGROUND

[0004] Thromboxane A2 (TXA2) is a prostanoid generated by the signal- activated metabolism of arachidonic acid by cyclooxygenase (COX) and downstream thromboxane synthase enzymes. Originally thought to be produced only in platelets where it potentiates their activation, TXA2 is now recognized to be produced in non-platelet tissue where it exerts a myriad of physiological and pathological effects through the activation of cellular thromboxane-prostanoid receptors. TXA2 rapidly and spontaneously degrades to TXB2, a stable prostanoid that circulates in the blood and is itself metabolized via two major oxidation and dehydrogenation pathways to numerous stable end-order metabolites (thromboxane B2 metabolites or TXB2-M) that are excreted by the kidney. Aspirin (ASA) exerts its major cardioprotective effect by blocking platelet TXA2 formation through the irreversible acetylation of COX-1. While effective at inhibiting platelet TXA2 generation in the vast majority of individuals, standard daily aspirin (ASA) therapy does not effectively suppress TXA2 generation in nucleated cells due to their capacity to regenerate COX- 1 or produce TXA2 through an inducible COX-2 pathway that is not inhibited by ASA. A number ofclinical studies in aspirin (ASA) users with cardiovascular disease (CVD) have found an association between urinary thromboxane B2 metabolites (TXB2-M) and risk of adverse cardiovascular (CV) events and mortality. It was recently demonstrated that urinary thromboxane B2 metabolites (TXB2-M) independently and robustly associated with long-term all-cause and cardiovascular (CV) mortality in a large unselected population irrespective of aspirin (ASA) use or presence of cardiovascular disease (CVD) (Rade JJ, Barton BA, Vasan RS, Kronsberg SS, Xanthakis V, Keaney JF, Jr., et al. Association of thromboxane generation with survival in aspirin users and nonusers. J Am Coll Cardiol 2022;80:233-50).

[0005] The most commonly used assay to measure urinary TXB2-M is an immunoassay utilizing a monoclonal primary antibody that predominantly recognizes the TXB2 metabolites 11-dehydro TXB2 and 1 l-dehydro-2,3-dinor TXB2 (Olson MT, Kickler TS, Lawson JA, McLean RC, Jani J, FitzGerald GA, Rade JJ. Effect of assay specificity on the association of urine 11 -dehydro thromboxane b2 determination with cardiovascular risk. J Thromb Haemost 2012; 10:2462-9). To account for differences in urine output and concentration, the raw thromboxane B2 metabolites (TXB2-M) concentration is adjusted / normalized to urine creatinine and expressed as pg / mg creatinine. A version of this immunoassay, the AspirinWorks® 1 l-dhTXB? Test Kit (Corgenix, Inc.), is United States Food and Drug Administration (FDA)-cleared for assessing aspirin (ASA) responsiveness and employs a dichotomous cut-point of 1500 pg / mg creatinine based on a study in 166 healthy individuals before and after aspirin (ASA) treatment (Geske FJ, Guyer KE, Ens G. Aspirinworks: A new immunologic diagnostic test for monitoring aspirin effect. Mol Diagn Ther 2008;12:51-4; Ames PR, Batuca JR, Muncy IJ, De La Torre IG, Pascoe-Gonzales S, Guyer K, et al. Aspirin insensitive thromboxane generation is associated with oxidative stress in type 2 diabetes mellitus. Thromb Res 2012;130:350-4). While some clinical studies in aspirin (ASA) users employed this 1500 pg / mg creatinine cut-point, larger studies tended to use study population-specific upper quartile values given that optimized cut-points for adverse clinical outcomes remain largely undefined (Kakouros N, Gluckman TJ, J.V. C, Kickler TS, Laws K, Barton.B.A., Rade JJ. Differential impact of serial measurment of nonplatelet thromboxane generation on long-term outcome after cardiac surgery. J Am Heart Assoc 2017;6:e007486; Rade JJ, Barton BA, Vasan RS, Kronsberg SS, Xanthakis V, Keaney JF, Jr., et al. Association of thromboxane generation with survival in aspirin users and nonusers. J Am Coll Cardiol 2022;80:233-50; Hariri E, Kakouros N, Bunsick DA, Russell SD, Mudd JO, Laws K, et al. Nonplatelet thromboxane generation is associated with impaired cardiovascular performance and mortality in heart failure. Am J Physiol Heart CircPhysiol 2022;323:H248-h55; Pastori D, Pignatelli P, Farcomeni A, Cangemi R, Hiatt WR, Bartimoccia S, et al. Urinary 11 -dehydro-thromboxane b2 is associated with cardiovascular events and mortality in patients with atrial fibrillation. Am Heart J 2015;170:490-7.el;Szczeklik W, Stodolkiewicz E, Rzeszutko M, Tomala M, Chrustowicz A, Zmudka K, Sanak M. Urinary 11 -dehydro-thromboxane b2 as a predictor of acute myocardial infarction outcomes: Results of leukotrienes and thromboxane in myocardial infarction (llimi) study. J Am Heart Assoc 2016;5).

[0006] Thromboxane B2 metabolites (TXB2-M) are filtered by the kidney and there is evidence that urine levels are proportional to renal function in both aspirin users and nonusers (Rade JJ, Barton BA, Vasan RS, Kronsberg SS, Xanthakis V, Keaney JF, Jr., et al.Association of thromboxane generation with survival in aspirin users and nonusers. J Am Coll Cardiol 2022;80:233-50; Kakouros N, Nazarian SM, Stadler PB, Kickler TS, Rade JJ. Risk factors for non-platelet thromboxane generation after coronary artery bypass graft surgery. Journal of the American Heart Association 2016;5:e002615). In populations where renal function is impaired or fluctuates, urinary thromboxane B2 metabolites (TXB2-M) adjusted only to urine creatinine may not accurately reflect systemic TXA2 generation and thus may alter its association with outcome risk.

[0007] Thus, a need exists to more accurately measure urinary thromboxane B2 metabolites (TXB2-M) and determine cut-point values for adverse cardiovascular events, cardiovascular mortality risk, stroke mortality risk, and long-term mortality risk.SUMMARY

[0008] The present disclosure provides methods of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample, the method comprising, consisting essentially or, or consisting of: determining the amount of thromboxane B2 metabolites (TXB2-M) in the urine sample, and adjusting or normalizing the amount of thromboxane B2 metabolites (TXB2-M) to creatinine (e.g., urine creatinine or creatinine level) in the urine sample (e.g., picogram (pg) of thromboxane B2 metabolites (TXB2-M) / milligram (mg) of creatinine).

[0009] In any aspect or embodiment described herein, (a) the amount of thromboxane B2 metabolites (TXB2-M) is determined by enzyme-linked immunosorbent assay (ELISA), mass spectrometry, western blot, nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, High / Ultra Performance Liquid Chromatography (H / UPLC), Liquid Chromatography Mass Spectrometer (LC-MS), Gas Chromatography Mass Spectrometer (GC-MS) technology, or a combination thereof; (b) the creatinine level is determined byenzyme-linked immunosorbent assay (ELISA), mass spectrometry, western blot, nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, High / Ultra Performance Liquid Chromatography (H / UPLC), Liquid Chromatography Mass Spectrometer (LC-MS), Gas Chromatography Mass Spectrometer (GC-MS) technology, or a combination thereof; or (c) a combination thereof.

[0010] In any aspect or embodiment described herein, the amount of thromboxane B2 metabolites (TXB2-M) adjusted or normalized to creatinine (e.g., pg / mg) in the urine sample is adjusted or normalized to renal function.

[0011] In any aspect or embodiment described herein, the renal function includes or is estimated glomerular filtration rate (eGFR) (e.g., TXB2-M / eGFR, such as pg-min / creatinine-mL- 1.73m2or a filtered prostanoid unit (FPU)).

[0012] In any aspect or embodiment described herein, the estimated glomerular filtration rate (eGFR) is determined by Chronic Kidney Disease Epidemiology Collaboration 2021 (CKD-EPI 2021) formula based on serum creatinine.

[0013] The present disclosure further provides methods of examining or determining mortality risk in a subject, the method comprising, consisting essentially of, or consisting of, performing the method of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample described herein, and determining or assigning the mortality risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) adjusted / normalized to creatinine level (e.g., pg / mg) in the urine sample.

[0014] In any aspect or embodiment described herein, a subject with a thromboxane B2 metabolites (TXB2-M) level of at least about 1291 pg / mg creatinine (e.g., about 1291 or greater pg / mg creatinine) for aspirin (ASA) users or at least about 5609 pg / mg creatinine (e.g., about 5609 or greater pg / mg creatinine) for non-aspirin (non- AS A) users is at increased mortality risk.

[0015] In any aspect or embodiment described herein, the subject is at increased mortality risk from a disease, disorder, or condition that includes or is at least one of cardiovascular disease, heart failure (e.g., heart failure with a preserved ejection fraction or heart failure with a reduced ejection fraction), stroke, or a combination thereof.

[0016] In any aspect or embodiment described herein, the disease, disorder, or condition is heart failure, and the subject has (1) two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria and two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) minor criteria, wherein at least one of: (i) each of the major criteria includes or is independently: paroxysmal nocturnal dyspnea or orthopnea; distendedneck veins (in other than the supine position); rales; increasing heart size by x-ray; acute pulmonary edema on chest x-ray; ventricular S3 gallop; increased venous pressure > 16 cm H2O; hepatojugular reflux; pulmonary edema, visceral congestion, and / or cardiomegaly shown on autopsy; weigh loss on heart failure treatment (e.g., 10 pounds / 5 days); or a combination thereof; (ii) each of the minor criteria includes or is independently bilaterial ankle edema, night cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion by x-ray, decrease in vital capacity by one-third from maximum record, tachycardia (e.g., > 120 beats per minute), pulmonary vascular engorgement on chest x-ray, or a combination thereof; or (iii) a combination thereof.

[0017] The present disclosure further provides methods of examining or determining mortality risk in a subject, the method comprising, consisting essentially of, or consisting of, performing the method of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample described herein, and determining or assigning the mortality risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) adjusted / normalized to creatinine level (e.g., pg / mg) in the urine sample, adjusted or normalized to renal function.

[0018] In any aspect or embodiment described herein, a subject with a TXB2- M / estimated glomerular filtration rate (eGFR) of about 16.6 pg-min / creatinine-mL- 1.73m2(e.g., about 16.6 or greater pg-min / creatinine-mL- 1.73m2) for aspirin (ASA) users or about 62.1 pg-min / creatinine-mL- 1.73m2(e.g., about 62.1 or greater pg-min / creatinine-mL- 1.73m2) for non-aspirin (ASA) users is at increased mortality risk.

[0019] In any aspect or embodiment described herein, the subject is at increased mortality risk from a disease, disorder, or condition that includes or is at least one of cardiovascular disease, heart failure (e.g., heart failure with a preserved ejection fraction or heart failure with a reduced ejection fraction), stroke, or a combination thereof.

[0020] In any aspect or embodiment described herein, the disease, disorder, or condition is hear failure, and the subject has (1) two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria and two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) minor criteria, wherein at least one of: (i) each of the major criteria includes or is independently: paroxysmal nocturnal dyspnea or orthopnea; distended neck veins (in other than the supine position); rales; increasing heart size by x-ray; acute pulmonary edema on chest x-ray; ventricular S3 gallop; increased venous pressure > 16 cm H2O; hepatojugular reflux; pulmonary edema, visceral congestion, and / or cardiomegaly shown on autopsy; weigh loss on heart failure treatment (e.g., 10 pounds / 5 days); or a combination thereof; (ii) each of the minor criteria includes or is independently bilaterialankle edema, night cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion by x-ray, decrease in vital capacity by one-third from maximum record, tachycardia (e.g., > 120 beats per minute), pulmonary vascular engorgement on chest x-ray, or a combination thereof; or (iii) a combination thereof.

[0021] The present disclosure additionally provides methods of examining or determining heart failure risk in a subject, the method comprising, consisting essentially of, or consisting of, performing the method of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample described herein, and determining or assigning the heart failure risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) adjusted / normalized to creatinine level (e.g., pg / mg) in the urine sample, adjusted or normalized to renal function.

[0022] In any aspect or embodiment described herein, the subject does not have heart failure.

[0023] In any aspect or embodiment described herein, the subject has no history of heart failure.

[0024] In any aspect or embodiment described herein, the subject has pre-heart failure (e.g., Stage A heart failure or Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure).

[0025] In any aspect or embodiment described herein, the subject has Stage A heart failure or Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure.

[0026] In any aspect or embodiment described herein, the subject has Stage A heart failure or Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure according to the 2022 AAC / AHA Stages of Heart Failure guidelines.

[0027] In any aspect or embodiment described herein, the subject has Stage A heart failure or Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure as described in Heidenreich PA, et al. 2022 AHA / ACC / HFS A Guideline for the Management of Heart Failure: A Report of the American College of Cardiology / American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 2022; 145(18):e895-el032.

[0028] In any aspect or embodiment described herein, the subject has Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure.

[0029] In any aspect or embodiment described herein, the subject has Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure according to the 2022 AAC / AHA Stages of Heart Failure guidelines.

[0030] In any aspect or embodiment described herein, the subject has Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure as described in Heidenreich PA, et al. 2022 AHA / ACC / HFSA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology / American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 2022; 145(18):e895-el032.

[0031] In any aspect or embodiment described herein, a subject with a TXB2- M / estimated glomerular filtration rate (eGFR) of about 16.6 pg-min / creatinine-mL- 1.73m2(e.g., about 16.6 or greater pg-min / creatinine-mL- 1.73m2) for aspirin (ASA) users or about 62.1 pg-min / creatinine mL- 1.73m2(e.g., about 62.1 or greater pg-min / creatinine mL- 1.73m2) for non-aspirin (ASA) users is at increased risk of developing heart failure.

[0032] In any aspect or embodiment described herein, the method further comprises treating the subject (e.g., treating the subject for the disease, disorder, or condition).

[0033] In any aspect or embodiment described herein, the disease, disorder, or condition is cardiovascular disease and the treatment includes or is at least one of a cholesterol lowering medication (e.g., statin, atorvastatin, simvastatin, rosuvastatin, pravastatin, fluvastatin, or a combination thereof), antiplatelet or blood thinning agent (e.g., aspirin, clopidogrel, rivaroxaban, ticagrelor, prasugrel, or a combination thereof), beta blocker (e.g., atenolol, bisoprolol, carvedilol, metoprolol, nebivolol, or a combination thereof), nitrate (e.g., hydralazine with nitrate), angiotensin-converting enzyme (ACE) inhibitor (e.g., ramipril, lisinopril, or a combination thereof), angiotensin-2 receptor blocker (such as candesartan, losartan, telmisartan, valsartan, or a combination thereof), calcium channel blocker (e.g., amlodipine, verapamil, diltiazem, or a combination thereof), diuretic (such as furosemide / frusemide, bumetanide, or a combination thereof), echocardiogram, angiogram, computed tomography (CT) scan, coronary angioplasty, (e.g., percutaneous coronary intervention (PCI), percutaneous transluminal coronary angioplasty (PTCA), or balloon angioplasty), coronary artery bypass grafting (CABG), heart transplant, omega-3 fatty acids, thromboxane-A synthase inhibitor (e.g., ridogrel, dazoxiben, imidazole, ozagrel, or a combination thereof), a thromboxane receptor antagonist (e.g., ridogrel, seratrodast, morniflumate, or a combination thereof), or a combination thereof.

[0034] In any aspect or embodiment described herein, the disease, disorder, or condition is at least one of heart failure (e.g., heart failure with a preserved ejection fraction or heart failure with a reduced ejection fraction) and the treatment includes or is heart failure medication, angiogram (e.g., percutaneous coronary intervention (PCI), percutaneous transluminal coronary angioplasty (PTCA), or balloon angioplasty), pacemaker, cardiac resynchronization therapy (CRT) device, implantable cardioverter defibrillator (ICD), a cardiac resynchronization therapy defibrillator (CRT-D), or a combination thereof.

[0035] In any aspect or embodiment described herein, the heart failure medication includes or is at least one of an antiplatelet or blood thinning agent (e.g., aspirin, clopidogrel, rivaroxaban, ticagrelor, prasugrel, or a combination thereof), ace inhibitor (e.g., ramipril, captopril, enalapril, lisinopril, perindopril, or a combination thereof), angiotensin-2 receptor blocker (e.g., candesartan, losartan, telmisartan, valsartan, or a combination thereof), beta blocker (e.g., atenolol, bisoprolol, carvedilol, metoprolol, nebivolol, or a combination thereof), mineralocorticoid receptor antagonist (e.g., spironolactone, eplerenone, or a combination thereof), diuretic (e.g., furosemide / frusemide, bumetanide, or a combination thereof), ivabradine, sacubitril valsartan, nitrate (e.g., hydralazine with nitrate), digoxin, SGLT2 inhibitor (e.g., empagliflozin, dapagliflozin, or a combination thereof), or combination thereof), thromboxane-A synthase inhibitor (e.g., ridogrel, dazoxiben, imidazole, ozagrel, or a combination thereof), a thromboxane receptor antagonist (e.g., ridogrel, seratrodast, morniflumate, or a combination thereof), glucagon-like peptide 1 (GLP- 1) receptor agonist (e.g., dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, tirzepatide, polyethylene glycol loxenatide, or a combination thereof), or a combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure. The drawings are only for the purpose of illustrating embodiments of the disclosure and are not to be construed as limiting the disclosure. Further objects, features, and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure.

[0037] Figures 1A and IB. (1A) Relationship between log-transformed urinary thromboxane B2 metabolites (TXB2-M) and estimated glomerular filtration rate eGFR in 1352aspirin (ASA) users and 1661 non-users enrolled in the Framingham Heart Study (FHS). (IB) Median filtered prostanoid unit in Framingham Heart Study (FHS) participants by aspirin (ASA) use. eGFR =estimated glomerular filtration rate; FPU =filtered prostanoid unit; TXB2- M =urinary thromboxane B2 metabolites (pg / mg creatinine).

[0038] Figure 2A and 2B. Comparative receiver-operator curves for the association of urinary TXB2-M and TXB2-M / eGFR to all-cause mortality in (2A) 1363 aspirin (ASA) users and (2B) 1680 aspirin (ASA) non-users in the primary study population. Area under the curve (AUC) is indicated for each cut-point.

[0039] Figures 3A, 3B, and 3C. Comparative long-term survival plots using optimized aspirin (ASA) use-specific TXB2-M and TXB2-M / eGFR cut-points. Kaplan-Meir survival plots for (3 A) 3013 Framingham Heart Study (FHS) participants and (3B) 105 patients with heart failure. (3C) Cumulative long-term incidence of cardiovascular / stroke mortality in 3013 Framingham Heart Study (FHS) participants. eGFR =estimated glomerular filtration rate; FPU =filtered prostanoid unit (pg-min / creatinine-mL- 1.73m2); TXB2-M =urinary thromboxane B2 metabolites (pg / mg creatinine).

[0040] Figure 4A. Mean urinaryTXB2-McFR (In FPU) in 2756 Framingham Heart Study Offspring / Omni participants stratified by ASA use and HF stage. Numbers of individuals in each category are shown. *P <0.04, #P <0.003 and **P <0.0001 compared to healthy group.

[0041] Figure 4B. Cumulative incidence and adjusted risk of HF in 2046 FHS participants with Stage B HF stratified by high and low urinary TXB2-MGFR based on ASA use-dependent cut-points (16.6 and 62.1 PFU for ASA users and non-users, respectively). Stage B HF was defined according to 2022 AHA / ACC / HFSA guidelines by the presence of any of the following baseline echocardiographic abnormalities: LVEF< 50%, global longitudinal strain <16%, lateral e’ <10 cm / s, average E / e’ >15, left atrial volume index >29 mL / m2, LV mass index >116 g / m2for men and >95 g / m2for women, relative wall thickness >0.42 or LV wall thickness >12 mm.DETAILED DESCRIPTION

[0042] The present inventors surprisingly and unexpectedly discovered that adjusting raw thromboxane B2 metabolites (TXB2-M) concentration for both the estimated glomerular filtration rate (eGFR) and urinary creatinine concentration), referred to herein as the filtered prostanoid unit (FPU, wherein 1 FPU =pg TXB2-M -min / urine creatinine-mL- 1.73m2), provided a more accurate marker for numerous adverse events. Optimal urinary TXB2-M andTXBi-M / eGFR dichotomous cut-point values for long-term mortality risk analysis were determined in a large, unselected population of aspirin (ASA) users and non-users who participated in the Framingham Heart Study (FHS) as well as in an external validation cohort of aspirin (ASA) users with heart failure (HF). The utility of these cut-point values was assessed for cardiovascular (CV) mortality risk and stroke mortality risk.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure.

[0044] Where a range of values is provided, it is understood that each intervening value in the range, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (for example, in the case of a group containing a number of carbon atoms in which case each carbon atom number falling within the range is provided), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either / or both of those included limits are also included in the disclosure.

[0045] It should also be understood that, in certain methods or processes described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0046] The following terms are used to describe the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

[0047] The articles "a" and "an" as used herein and in the appended claims are used herein to refer to one or to more than one (that is, to at least one or one or more of) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element, unless otherwise indicated.

[0048] The phrase “and / or”, as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that areconjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or’- should be construed in the same fashion, that is, “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0049] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (that is, “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0050] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0051] In the claims, as well as in the specification above, all transitional phrases such as “comprising,’" “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended (that is, to mean including but not limited to). It is expressly contemplated that all embodiments, and claims reciting one of the open-ended transitional phrases can be written with any other transitional phrase, which may be more limiting, unless clearly precluded by the context or art. Only the transitional phrases “consisting of’ and “consisting essentially of” shall be closed or semiclosed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0052] Method of Detecting Thromboxane B2 metabolites (TXB2-M)

[0053] The present disclosure provides methods of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample, the method comprising, consisting essentially or, or consisting of: determining the amount of thromboxane B2 metabolites (TXB2-M) in the urine sample, and adjusting or normalizing the amount of thromboxane B2 metabolites (TXB2-M) to creatinine (e.g., urine creatinine or creatinine level) in the urine sample (e.g., picogram (pg) of thromboxane B2 metabolites (TXB2-M) / milligram (mg) of creatinine).

[0054] In any aspect or embodiment described herein, the amount of thromboxane B2 metabolites (TXB2-M) is determined by enzyme-linked immunosorbent assay (ELISA), mass spectrometry, western blot, nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, High / Ultra Performance Liquid Chromatography (H / UPLC), Liquid Chromatography Mass Spectrometer (LC-MS), Gas Chromatography Mass Spectrometer (GC-MS) technology, or a combination thereof. In any aspect or embodiment described herein, the creatinine level is determined by enzyme-linked immunosorbent assay (ELISA), mass spectrometry, western blot, nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, High / Ultra Performance Liquid Chromatography (H / UPLC), Liquid Chromatography Mass Spectrometer (LC-MS), Gas Chromatography Mass Spectrometer (GC-MS) technology, or a combination thereof.

[0055] In any aspect or embodiment described herein, the amount of thromboxane B? metabolites (TXB2-M) adjusted or normalized to creatinine level (e.g., pg / mg) in the urine sample is adjusted or normalized to renal function.

[0056] In any aspect or embodiment described herein, the renal function includes or is estimated glomerular filtration rate (eGFR) (e.g., TXB2-M / eGFR, such as pg-min / creatinine-mL- 1.73m2or a filtered prostanoid unit (FPU)).

[0057] In any aspect or embodiment described herein, the estimated glomerular filtration rate (eGFR) is determined by Chronic Kidney Disease Epidemiology Collaboration 2021 (CKD-EPI 2021) formula based on serum creatinine.

[0058] Method of Examining, Determining, and / or Treating Mortality Risk and / or Heart Failure Risk

[0059] The present disclosure further provides methods of examining or determining mortality risk in a subject, the method comprising, consisting essentially of, or consisting of, performing the method of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample described herein, and determining or assigning the mortality risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) adjusted / normalized to creatinine level (e.g., pg / mg) in the urine sample. For example, in any aspect or embodiment described herein, determining or assigning the mortality risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) adjusted / normalized to creatinine level (e.g., pg / mg) in the urine sample, is performed as discussed in the examples.

[0060] In any aspect or embodiment described herein, a subject with a thromboxane B2 metabolites (TXB2-M) level of at least about 1291 pg / mg creatinine (e.g., about 1291 or greater pg / mg creatinine) for aspirin (ASA) users or at least about 5609 pg / mg creatinine (e.g., about 5609 or greater pg / mg creatinine) for non- aspirin (non- AS A) users is at increased mortality risk.

[0061] In any aspect or embodiment described herein, the subject is at increased mortality risk from a disease, disorder, or condition that includes or is at least one of cardiovascular disease, heart failure (e.g., heart failure with a preserved ejection fraction or heart failure with a reduced ejection fraction), stroke, or a combination thereof.

[0062] In any aspect or embodiment described herein, the disease, disorder, or condition is heart failure, and the subject has (1) two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria and two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) minor criteria, wherein at least one of: (i) each of the major criteria includes or is independently: paroxysmal nocturnal dyspnea or orthopnea; distended neck veins (in other than the supine position); rales; increasing heart size by x-ray; acute pulmonary edema on chest x-ray; ventricular S3 gallop; increased venous pressure > 16 cm H2O; hepatojugular reflux; pulmonary edema, visceral congestion, and / or cardiomegaly shown on autopsy; weigh loss on heart failure treatment (e.g., 10 pounds / 5 days); or a combination thereof; (ii) each of the minor criteria includes or is independently bilaterial ankle edema, night cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion byx-ray, decrease in vital capacity by one-third from maximum record, tachycardia (e.g., > 120 beats per minute), pulmonary vascular engorgement on chest x-ray, or a combination thereof; or (iii) a combination thereof.

[0063] The present disclosure further provides methods of examining or determining mortality risk in a subject, the method comprising performing the method of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample described herein, and determining or assigning the mortality risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) adjusted / normalized to creatinine level (e.g., pg / mg) in the urine sample, adjusted or normalized to renal function. For example, in any aspect or embodiment described herein, determining or assigning the mortality risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) adjusted / normalized to creatinine level (e.g., pg / mg) in the urine sample, adjusted or normalized to renal function, is performed as discussed in the examples.

[0064] In any aspect or embodiment described herein, a subject with a TXB2- M / estimated glomerular filtration rate (eGFR) of about 16.6 pg-min / creatinine-mL- 1.73m2(e.g., about 16.6 or greater pg-min / creatinine-mL- 1.73m2) for aspirin (ASA) users or about 62.1 pg-min / creatinine-mL- 1.73m2(e.g., about 62.1 or greater pg-min / creatinine-mL- 1.73m2) for non-aspirin (ASA) users is at increased mortality risk.

[0065] In any aspect or embodiment described herein, the subject is at increased mortality risk from a disease, disorder, or condition that includes or is cardiovascular disease, heart failure (e.g., heart failure with a preserved ejection fraction or heart failure with a reduced ejection fraction), stroke, or a combination thereof.

[0066] In any aspect or embodiment described herein, the disease, disorder, or condition is heart failure, and the subject has (1) two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria and two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) minor criteria, wherein: (i) each of the major criteria includes or is independently: paroxysmal nocturnal dyspnea or orthopnea; distended neck veins (in other than the supine position); rales; increasing heart size by x-ray; acute pulmonary edema on chest x-ray; ventricular S3 gallop; increased venous pressure > 16 cm H2O; hepatojugular reflux; pulmonary edema, visceral congestion, and / or cardiomegaly shown on autopsy; weigh loss on heart failure treatment (e.g., 10 pounds / 5 days); or a combination thereof; (ii) each of the minor criteria includes or is independently bilaterial ankle edema, night cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion by x-ray, decrease in vital capacity byone-third from maximum record, tachycardia (e.g., > 120 beats per minute), pulmonary vascular engorgement on chest x-ray, or a combination thereof; or (iii) a combination thereof.

[0067] The present disclosure additionally provides methods of examining or determining heart failure risk in a subject, the method comprising, consisting essentially of, or consisting of, performing the method of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample described herein, and determining or assigning the heart failure risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) adjusted / normalized to creatinine level (e.g., pg / mg) in the urine sample, adjusted or normalized to renal function.

[0068] In any aspect or embodiment described herein, the subject has no history of heart failure. For example, in any aspect or embodiment described herein, the subject does not have heart failure.

[0069] In any aspect or embodiment described herein, the subject has pre-heart failure (e.g., Stage A heart failure or Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure).

[0070] In any aspect or embodiment described herein, the subject has Stage A heart failure or Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure.

[0071] In any aspect or embodiment described herein, the subject has Stage A heart failure or Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure according to the 2022 AAC / AHA Stages of Heart Failure guidelines.

[0072] In any aspect or embodiment described herein, the subject has Stage A heart failure or Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure as described in Heidenreich PA, et al. 2022 AHA / ACC / HFS A Guideline for the Management of Heart Failure: A Report of the American College of Cardiology / American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 2022; 145(18):e895-el032.

[0073] In any aspect or embodiment described herein, the subject has Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure.

[0074] In any aspect or embodiment described herein, the subject has Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure according to the 2022 AAC / AHA Stages of Heart Failure guidelines.

[0075] In any aspect or embodiment described herein, the subject has Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure as described in Heidenreich PA, et al. 2022 AHA / ACC / HFSA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology / American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 2022; 145(18):e895-el032.

[0076] In any aspect or embodiment described herein, a subject with a TXB2- M / estimated glomerular filtration rate (eGFR) of about 16.6 pg-min / creatinine-mL- 1 .73m2(e.g., about 16.6 or greater pg-min / creatinine-mL- 1.73m2) for aspirin (ASA) users or about 62.1 pg-min / creatinine mL- 1.73m2(e.g., about 62.1 or greater pg-min / creatinine mL- 1.73m2) for non-aspirin (ASA) users is at increased risk of developing heart failure.

[0077] In any aspect or embodiment described herein, the method further comprises treating the subject (e.g., treating the subject for the disease, disorder, or condition).

[0078] In any aspect or embodiment described herein, the disease, disorder, or condition is cardiovascular disease and the treatment includes or is at least one of a cholesterol lowering medication (e.g., statin, atorvastatin, simvastatin, rosuvastatin, pravastatin, fluvastatin, or a combination thereof), antiplatelet or blood thinning agent (e.g., aspirin, clopidogrel, rivaroxaban, ticagrelor, prasugrel, or a combination thereof), beta blocker (e.g., atenolol, bisoprolol, carvedilol, metoprolol, nebivolol, or a combination thereof), nitrate (e.g., hydralazine with nitrate), angiotensin-converting enzyme (ACE) inhibitor (e.g., ramipril, lisinopril, or a combination thereof), angiotensin-2 receptor blocker (such as candesartan, losartan, telmisartan, valsartan, or a combination thereof), calcium channel blocker (e.g., amlodipine, verapamil, diltiazem, or a combination thereof), diuretic (such as furosemide / frusemide, bumetanide, or a combination thereof), echocardiogram, angiogram, computed tomography (CT) scan, coronary angioplasty, (e.g., percutaneous coronary intervention (PCI), percutaneous transluminal coronary angioplasty (PTCA), or balloon angioplasty), coronary artery bypass grafting (CABG), heart transplant, omega-3 fatty acids, thromboxane-A synthase inhibitor (e.g., ridogrel, dazoxiben, imidazole, ozagrel, or a combination thereof), a thromboxane receptor antagonist (e.g., ridogrel, seratrodast, morniflumate, or a combination thereof), or a combination thereof.

[0079] In any aspect or embodiment described herein, the disease, disorder, or condition is heart failure (e.g., heart failure with a preserved ejection fraction or heart failure with a reduced ejection fraction) and the treatment includes or is at least one of heart failure medication, angiogram (e.g., percutaneous coronary intervention (PCI), percutaneoustransluminal coronary angioplasty (PTCA), or balloon angioplasty), pacemaker, cardiac resynchronization therapy (CRT) device, implantable cardioverter defibrillator (ICD), a cardiac resynchronization therapy defibrillator (CRT-D), or a combination thereof.

[0080] In any aspect or embodiment described herein, the heart failure medication includes or is at least one of an antiplatelet or blood thinning agent (e.g., aspirin, clopidogrel, rivaroxaban, ticagrelor, prasugrel, or a combination thereof), ace inhibitor (e.g., ramipril, captopril, enalapril, lisinopril, perindopril, or a combination thereof), angiotensin-2 receptor blocker (e.g., candesartan, losartan, telmisartan, valsartan, or a combination thereof), beta blocker (e.g., atenolol, bisoprolol, carvedilol, metoprolol, nebivolol, or a combination thereof), mineralocorticoid receptor antagonist (e.g., spironolactone, eplerenone, or a combination thereof), diuretic (e.g., furosemide / frusemide, bumetanide, or a combination thereof), ivabradine, sacubitril valsartan, nitrate (e.g., hydralazine with nitrate), digoxin, SGLT2 inhibitor (e.g., empagliflozin, dapagliflozin, or a combination thereof), or combination thereof), thromboxane-A synthase inhibitor (e.g., ridogrel, dazoxiben, imidazole, ozagrel, or a combination thereof), a thromboxane receptor antagonist (e.g., ridogrel, seratrodast, morniflumate, or a combination thereof), glucagon-like peptide 1 (GLP- 1) receptor agonist (e.g., dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, tirzepatide, polyethylene glycol loxenatide, or a combination thereof), or a combination thereof.

[0081] Abbreviations: TXB2-M =thromboxane B2 metabolites; ASA =aspirin; eGFR =estimated glomerular filtration rate; RMST restricted mean survival time; AIC =Akaike Information Criterion; AUC =area under the curve; TXA2 ^thromboxane A2; COX cyclooxygenase; CVD =cardiovascular disease; CV cardiovascular; ELISA enzyme- linked immunosorbent assay; FDA =United States Food and Drug Administration; pg =picogram; mg =milligram; FPU =filtered prostanoid unit; FHS =Framingham Heart Study; HF =heart failure; mL =milliliter; IQR =inter-quartile range; CI =Confidence interval; HR =hazard ratio; OR =odds ratio; ROC receiver-operator curve.EXAMPLES

[0082] Systemic thromboxane A2 generation, assessed by quantifying the concentration of stable thromboxane B2 metabolites (TXB2-M) in the urine adjusted for urinary creatinine, is strongly associated with mortality risk. The following experiments examine the optimal thromboxane B2 metabolites (TXB2-M) cut-points for aspirin (ASA) users and non-users, and whether adjusting thromboxane B2 metabolites (TXB2-M) forestimated glomerular filtration rate (eGFR) in addition to urine creatinine improves mortality risk assessment. Urinary thromboxane B2 metabolites (TXB2-M) were measured by competitive enzyme-linked immunosorbent assay (ELISA) in 1363 aspirin (ASA) users and 1681 non-users participating in the Framingham Heart Study (FHS). Optimal cut-points were determined for thromboxane B2 metabolites (TXB2-M) and TXB2-M / eGFR using log-rank statistics and used to assess mortality risk by Cox proportional hazard modeling and restricted mean survival time (RMST). Multivariable models were compared using Akaike Information Criterion (AIC). A cohort of 105 aspirin (ASA) users with heart failure was used for external validation. Optimized cut-points of thromboxane B2 metabolites (TXB2-M) were 1291 and 5609 pg / mg creatinine and of TXB2-M / eGFR were 16.6 and 62.1 filtered prostanoid units (defined as pg-min / creatinine-mL- 1.73m2), for aspirin (ASA) users and non-users, respectively. TXB2-M / eGFR cut-points provided more robust all-cause mortality risk discrimination than TXB2-M cut-points with a larger unadjusted hazard ratio (2.88 versus 2.16, AIC P<0.0001) and greater differences in restricted mean survival time (RMST) between exposure groups (1.46 versus 1.10 years), findings that were confirmed in the external validation cohort of aspirin (ASA)-users. TXB2-M / eGFR cut-points also provided better cardiovascular / stroke mortality risk discrimination than TXB2-M cut-points (unadjusted hazard ratio 3.31 versus 2.13, AIC P<0.0001). Adjustment for eGFR strengthens the association of urinary TXB2-M with long-term mortality risk irrespective of aspirin (ASA) use.METHODS

[0083] The primary study population consisted of 3044 participants in the Framingham Heart Study (FHS) Offspring and Omni Cohorts who attended Exam cycle 8 (2005-2008) and Exam cycle 3 (2007 to 2008), respectively, in whom TXB -M were measured in available urine samples banked at the time of the examination. The characteristics of these participants at the time of the index examinations and the relationship of urinary TXB2-M to long-term survival stratified by aspirin (ASA) use have previously been reported (Rade JJ, Barton BA, Vasan RS, Kronsberg SS, Xanthakis V, Keaney JF, Jr., et al. Association of thromboxane generation with survival in aspirin users and nonusers. J Am Coll Cardiol 2022;80:233-50.). All Framingham Heart Study (FHS) study participants are under continuous surveillance for cardiovascular events. Survival data were available in 3043 (99.9%) participants with the cause of death adjudicated by a group of three experienced investigators after review of the medical records (D'Agostino RB, Sr., Vasan RS, Pencina MJ, Wolf PA, Cobain M, Massaro JM, Kannel WB. General cardiovascular risk profile for use in primary care: The framingham heart study. Circulation 2008; 117:743-53). The external validation population consisted of 105 patients on daily aspirin (ASA) therapy who underwent clinically indicted invasive right heart catheterization for the evaluation of heart failure (HF), valvular heart disease or after cardiac transplantation at the Johns Hopkins Hospital (2010-2011) and the UMass Memorial Medical Center (2018-2019) in whom urinary TXB2-M were measured at the time of the cardiac catheterization (Hariri E, Kakouros N, Bunsick DA, Russell SD, Mudd JO, Laws K, et al. Nonplatelet thromboxane generation is associated with impaired cardiovascular performance and mortality in heart failure. Am J Physiol Heart Circ Physiol 2022;323:H248-h55). Written informed consent was obtained from participants in both study populations prior to their participation and protocols were approved by the human subject institutional review boards of the Boston University School of Medicine (primary study cohort), the University of Massachusetts Chan Medical School (primary and validation study cohorts) and the Johns Hopkins School of Medicine (validation study cohort).

[0084] TXB2-M was measured in banked urine samples stored at -80°C in duplicate using the AspirinWorks® HdhTXB2 Test Kit (Corgenix Inc., Bromfield, CO) according to the manufacturer’s instruction and normalized to urine creatinine. This competitive ELISA has a limit of detection of 156.25 pg / mL with a linear range up to 5000 pg / mL. For statistical purposes, the 29 samples (0.95%) with values below the limit of detection were reported as 156.3 pg / mL. Samples >5000 pg / mL were re-assayed at an additional 1:4 dilution and the 35(1.1%) that remained above the linear range of the assay at this higher dilution were reported as 20,000 pg / mL for statistical purposes. The intra-assay coefficient of variance was 3.3%. The stability of TXB2-M measurements with this assay in urine after long-term storage and multiple freeze-thaw cycles was previously verified in our laboratory (Olson MT, Kickler TS, Lawson JA, McLean RC, Jani J, FitzGerald GA, Rade JJ. Effect of assay specificity on the association of urine 11 -dehydro thromboxane b2 determination with cardiovascular risk. J Thromb Haemost 2012;10:2462-9). Urine creatinine was measured as previously described (Keaney JF, Jr., Larson MG, Vasan RS, Wilson PW, Lipinska I, Corey D, et al. Obesity and systemic oxidative stress: Clinical correlates of oxidative stress in the framingham study. Arterioscler Thromb Vase Biol 2003;23:434-9). eGFR was calculated using the Chronic Kidney Disease Epidemiologic Collaboration 2021 (CKD-EPI 2021) formula based on serum creatinine measured in Framingham Heart Study (FHS) participants at the time of index examinations and in patients at the time of right heart catheterization as described (Inker LA, Eneanya ND, Coresh J, Tighiouart H, Wang D, Sang Y, et al. New creatinine- and cystatin c- based equations to estimate gfr without race. N Engl J Med 2021 ;385: 1737-49).

[0085] Optimized cut-point values for urinary TXB2-M expressed as pg / mg urine creatinine and TXB2-M / eGFR expressed as FPU (1FPU = pg-min / urine creatinine-mL- 1.73m2) for the outcome of all-cause long-term mortality were defined for aspirin (ASA) users and non-users in the primary study population using the method of Cental and O’ Quigley based on log-rank statistics and confirmed with ROC analysis to determine the maximum Youden index (Cental C, O'Quigley J. An application of changepoint methods in studying the effect of age on survival in breast cancer.Computational Statistics and Data Analysis 1999;30:253-70; Youden WJ. Index for rating diagnostic tests. Cancer 1950;3:32-5). The relative ability of these cut-points to assess mortality risk was evaluated using Cox proportional hazards models with k-fold internal cross-validation (k=10) and reported as the averaged hazard ratio and 95% confidence interval (CI) separately for aspirin (ASA) users and non-users (Jung Y, Hu J. A k-fold averaging cross-validation procedure. J Nonparametr Stat 2015;27:167-79). Akaike Information Criterion (AIC) was used to compare the relative strength of models employing the different cut-points (Akaike N. Information theory and an extension of the maximum liklihood principle. In: Petrov BN, Csaki F, editors. Second international symposium on information theory Budapest; 1973). Restricted mean survival time (RMST) and multivariable Cox proportional hazard modeling were additionally used to further explore the relationship of urinary TXB2-M and TXB2-M / eGFR to time to death in both the primary(combined aspirin (ASA) users and non-users) and external validation (aspirin (ASA) users only) cohorts and to make comparisons between optimized and alternative cut-points used in the original studies. All statistical analyses were performed using SAS 9.4 software (SAS Institute, Inc., Cary, NC).RESULTS

[0086] The primary study cohort consisted of 3044 participants in the Framingham Heart Study (FHS) Offspring and Omni Cohorts who attended Exam cycles 8 and 3, respectively. The clinical characteristics of the 1363 (44.8%) participants taking aspirin (ASA) compared to the 1681 (55.2%) participants not taking aspirin (ASA) at the index examination are shown in Table 1. The median eGFR calculated using the Chronic Kidney Disease Epidemiologic Collaboration 2021 (CKD-EPI 2021) formal was slightly lower in aspirin (ASA) users as compared to non-users (82 mL / min / 1.73m2, IQR 69-93, vs. 87 mL / min / 1.73m2, IQR, 75-97, P <0.0001). There were significant correlations between eGFR and urinary TXB2-M observed in both aspirin (ASA) users than non-users (Figure 1A). The median urinary TXfb-M / eGFR ratio was about 70% lower in aspirin (ASA) users compared to non-users (Figure IB), consistent with the ability of aspirin (ASA) to suppress platelet and incompletely suppress non-platelet TXA2 generation.Table 1. Clinical and laboratory characteristics of Framingham Heart Study participants at time of the index examination stratified by aspirin use (Patrono C, Ciabattoni G, Patrignani P.Human eicosanoid biosynthesis and metabolism. Mt Sinai J Med 1992;59:157-62).Abbreviations for Table 1 : SD, standard deviation; BMI, body mass index; eGFR, estimated glomerular filtration rate; LVEF, left ventricular ejection fraction; ECG, electrocardiogram; PCI, percutaneous coronary intervention; CABG, coronary artery bypass graft; COPD, chronic obstructive pulmonary disease; DVT, deep vein thrombosis; PE, pulmonary embolus; ACEi, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; LDL, low-density lipoprotein; HDL, high-density lipoprotein; CRP, C-reactive protein; MCP, macrophage chemotactic factor; IL-6, interleukin-6; Lp-PLA2, lipoprotein-associated phospholipase A2; IQR, interquartile range.

[0087] Optimal dichotomous cut-points of urinary TXB2-M and TXBz-M / eGFR were determined separately for 1363 aspirin (ASA) users and 1680 non-users for the outcome of all-cause mortality over a median observation period of 13.0 (IQR, 11.8-13.7) years. Table 2 shows the optimized cut-points and the 4thquartile cut-points used in the original study, along with their associated mean hazard ratios (HR). The comparative receiver-operator curves for aspirin (ASA) users and non-users are shown in Figures 2A and 2B. As aspirin (ASA) usedependent TXB2-M cut-points can be used for survival analyses in mixed populations of aspirin (ASA) users and non-users, comparative outcome risk analyses was performed in the subgroup of 3013 Framingham Heart Study (FHS) participants in whom eGFR was calculable.Table 2. Optimized urinary TXB2-M and TXBz-M / eGFR cut-points for all-cause mortality risk in Framingham Heart Study participants stratified by aspirin (ASA) use.*N=1352 and 1661 for aspirin (ASA) users and non-users, respectively.#To convert to from pg / mg creatinine to pg / pmol creatinine divide by 8.842.Abbreviations for Table 2: ASA, aspirin; CI, confidence interval; eGFR, estimated glomerular filtration rate; FPU =filtered prostanoid unit (1 FPU =pg TXBi-M-min / urine creatinine-mLT.73nr); HR, hazard ratio; N / A, not applicable; TXB2-M, thromboxane B2 metabolites.

[0088] The comparative survival plots using the different cut-points are shown in Figure 3A. Use of optimized TXEU-M / eGFR cut-points resulted in a higher unadjusted mortality hazard ratio between exposure groups and significantly better model fit by AIC than use of optimized TXB2-M or the 4thquartile cut-points used in the original study (Table 3), a finding that persisted after adjustment of age and sex (Table 4). As a sensitivity analysis, restricted mean survival times (RMST) between exposure groups were determined and revealed that use of the optimized TXBs-M / eGFR cut-points resulted in a larger difference in restricted mean survival time (RMST) between exposure groups than use of optimized TXB2-M or 4thquartile cut-points (Table 3). the use of the optimized TXB2- M / eGFR cut-point was externally validated in 105 aspirin (ASA) users undergoing invasive hemodynamic assessment for the evaluation of heart failure (HF), valvular heart disease or after cardiac transplantation where urinary TXB2-M was associated with both indices of cardiac performance and all-cause mortality. (19) The clinical characteristics of these participants are shown in Table 5. Figure 3B shows the comparative survival plots and Table 6 shows the hazard ratios and restricted mean survival time (RMST) for all-cause mortality between exposure groups over a median observation period of 3.8 (IQR 1.7, 7.0) years. Compared to the 1500 pg / mg creatinine cut-point for aspirin (ASA) users established by the manufacturer of the assay and used in the original study, use of the optimized TXB2-M / eGFR cut-point resulted in better risk stratification between exposure groups, even when adjusted for age and sex.Table 3. Relative differences in all-cause mortality risk and survival times in a cohort of 3013 Framingham Heart Study participants using different aspirin (ASA) use-dependent urinaryTXBi-M cut-points.*P <0.0001 versus AIC for 4thquartile cut-point model.#P <0.0001 versus AIC for optimized TXB2-M and 4thquartile cut-point models.fTo convert to from pg / mg creatinine to pg / pmol creatinine divide by 8.842.Abbreviations for Table 3: AIC, Akaike Information Criterion; ASA, aspirin; CI, confidence interval; FPU, filtered prostanoid unit (1 FPU =pg-min / creatinine-mL- 1.73m2); HR, hazard ratio; RMST, restricted mean survival time; TXB2-M, thromboxane B 2 metabolites.Table 4. Relative differences in all-cause mortality risk adjusted for age and sex in a cohort of 3013 Framingham Heart Study participants using optimized aspirin (ASA) use-dependent optimized and 4thquartile cut-points*Adjusted for age and sex.#P <0.0001 versus AIC for optimized TXB2-M and 4thquartile cut-point models. fTo convert pg / mg creatinine to pg / pmol creatinine divide by 8.842.Abbreviations for Table 4: AIC, Akaike Information Criterion; ASA, aspirin; CI, confidence interval; FPU, filtered prostanoid unit (1 FPU =pg-min / creatinine-mL-1.73m2); HR, hazard ratio; RMST, restricted mean survival time; TXB2-M, thromboxane B 2 metabolites.Table 5. Baseline characteristics of the 105 aspirin (ASA) users in the validation population stratified by low and high non-platelet TXA2 generation (Huang JS, Ramamurthy SK, Lin X,Le Breton GC. Cell signalling through thromboxane a2 receptors. Cell Signal 2004; 16:521- 33).* Pre-transplant characteristics.# Left ventricular assist device subjects (n=5) excluded. tTo convert creatinine from mg / dL to pmol / L multiply by 88.42.Abbreviations for Table 5: SD, standard deviation; IQR, interquartile range; BMI, body mass index; HFrEF, heart failure with reduced ejection fraction; ICD, internal cardiac defibrillator; LVEF, left ventricular ejection fraction; ACEi, angiotensin-converting enzyme inhibitor;ARB, angiotensin receptor blocker; NSAID, non-steroidal anti-inflammatory drug.Table 6. Relative differences in all-cause mortality risk and survival time in a cohort of 105 heart failure patients using different cut-points for aspirin (ASA) users.*Adjusted for age and sex.#To convert to from pg / mg creatinine to pg / pmol creatinine divide by 8.842.Abbreviations for Table 6: ASA, aspirin; HR, hazard ratio; CI, confidence interval; FPU, filtered prostanoid unit (1 FPU =pg-min / creatinine-mL- 1.73m2); RMST, restricted mean survival time; TXB2-M, thromboxane B2 metabolites.

[0089] The use of the optimized TXEL-M / eGFR cut-points for all-cause mortality to improve risk stratification for cardiovascular (CV) / stroke mortality was examined. Figure 3C shows the comparative cumulative incidence plots for cardiovascular (CV) / stroke death in 3013 Framingham Heart Study (FHS) participants using the different cut-points. Like allcause mortality, the use of the optimized TXEF-M / eGFR cut-points resulted in improved risk stratification for cardiovascular (CV) / stroke mortality risk compared to optimized TXB2-M or 4thquartile cut-points as evidenced by a greater HR, significantly lower AIC, and greater difference in restricted mean survival time (RMST) between exposure groups (Table 7 and Table 8).Table 7. Relative cardiovascular and stroke mortality risk and survival times in a cohort of3013 Framingham Heart Study participants using different aspirin (ASA) use-dependent urinary TXB2-M cut-points.*P =0.03 versus AIC for 4thquartile cut-point model.#P <0.0001 versus AIC for optimized TXB2-M and 4thquartile cut-point models.Abbreviations for Table 7: AIC, Akaike Information Criterion; ASA, aspirin; CI, confidence interval; FPU, filtered prostanoid unit (1 FPU =pg-min / creatinine-mL- 1.73m2); HR, hazard ratio; RMST, restricted mean survival time; TXB2-M, thromboxane B2 metabolites.Table 8. Relative differences in adjusted cardiovascular / stroke mortality risk in a cohort of 3013 Framingham Heart Study participants using optimized urinary TXB2-M cut-points for aspirin (ASA) users and non-users.*Adjusted for age and sex.#P <0.003 versus AIC for optimized TXB2-M and 4thquartile cut-point models. fTo convert pg / mg creatinine to pg / pmol creatinine divide by 8.842.Abbreviations for Table 8: AIC, Akaike Information Criterion; ASA, aspirin; CI, confidence interval; eGFR, estimated glomerular filtration rate; FPU, filtered prostanoid unit (1 FPU =pg-min / creatinine-mL- 1.73m2); HR, hazard ratio; TXB2-M, thromboxane B2 metabolites.

[0090] TXB2-MGFR identifies high-risk pre-Heart Failure. Heart failure (HF) is a multisystem disease that progresses through recognized stages. Stage A is comprised of individuals without HF, but with HF risk factors (cardiovascular (CV) risk factors, genetic predisposition, etc.). Stage B is comprised of those with subclinical cardiac structural abnormalities or evidence of injury / stress (elevated cardiac troponin or N-terminal Pro-Brain Natriuretic Peptide (NT-proBNP)). Stages C and D are denoted by overt and end-stage HF, respectively.

[0091] Figure 4A shows the mean urinary TXB2-MGFR (In FPU) in 2756 Framingham Heart Study Offspring / Omni participants stratified by ASA use and HF stage. Numbers of individuals in each category are shown. *P <0.04, #P <0.003 and **P <0.0001 compared to healthy group. Figure 4B shows the cumulative incidence and adjusted risk of HF in 2046 FHS participants with Stage B HF stratified by high and low urinary TXB2-MGFR based on ASA use-dependent cut-points (16.6 and 62.1 PFU for ASA users and non-users, respectively). Stage B HF was defined according to 2022 AHA / ACC / HFSA guidelines by the presence of any of the following baseline echocardiographic abnormalities: LVEF< 50%, global longitudinal strain <16%, lateral e’ < 10 cm / s, average E / e’ >15, left atrial volume index >29 mL / nf, LV mass index >116 g / m2for men and >95 g / m2for women, relative wall thickness >0.42 or LV wall thickness >12 mm.

[0092] In the analysis of 2756 FHS Offspring / Omni participants, mean urinary TXB2- MGFR was observed to be proportional to HF stage (Figure 4A). Furthermore, while urinary TXB2-MGF further stratified HF risk in healthy and stage A individuals, urinary TXB2-MGFR was able to identify stage B individuals at particularly high risk of developing HF (Figure 4B).

[0093] DISCUSSION

[0094] The examples herein determine optimized cut-off values for urinary TXB2-M and TXB2-M / eGFR for the outcome of all-cause mortality in both aspirin (ASA) users and non-users, wherein the optimized TXB2-M / eGFR cut-points provided better prognostic information than optimized TXB2-M, population-defined 4thquartile or manufacturer-defined cut-points for all-cause and CV / stroke mortality outcome analyses.

[0095] Systemic TXA2generation is a novel and potent mortality risk factor that is readily assessed by measuring its stable downstream metabolites in the urine. The AspirinWorks® 1 ldhTXB2Test Kit is currently the only clinically available assay formeasuring urinary TXB2-M (Geske FJ, Guyer KE, Ens G. Aspirinworks: A new immunologic diagnostic test for monitoring aspirin effect. Mol Diagn Ther 2008;12:51-4). As this assay was designed and United States Food and Drug Administration (FDA) cleared to assess aspirin (ASA)-responsiveness, it is understandable that the manufacturer-defined cutpoint of 1500 pg / mg creatinine cut-point may not be ideal for outcome analyses and therefore, most clinical studies have relied on population-specific dichotomous upper quartile cut-off values for outcome analyses. Only one prior study involving 449 aspirin (ASA) users with cardiovascular (CV) disease defined an optimal cut-point for mortality risk using this assay and found that individuals with urinary TXB2-M >1598 pg / mg creatinine had a significantly higher 5 -year risk of cardiovascular death compared to those with lower values (OR 2.86, CI 1.58, 5.19, P =0.001) (Vasudevan A, Tecson KM, Bennett-Firmin J, Bottiglieri T, Lopez LR, Peterson M, et al. Prognostic value of urinary 1 1 -dehydro-thromboxane b(2) for mortality: A cohort study of stable coronary artery disease patients treated with aspirin. Catheter Cardiovasc Interv 2018;92:653-8). In the large, unselected population examined here, a slightly lower optimized cut-point (1291 pg / mg creatinine) was found for all-cause mortality in aspirin (ASA) users that could also be used for cardiovascular (CV) / stroke mortality analysis (Table 7). Systemic TXA2 generation has recently been recognized to be an even more robust mortality risk factor in individuals not taking aspirin (ASA) and the optimal cut-off value (5609 pg / mg creatinine) in this group was determined for the first time here. It was further confirmed that these optimized aspirin (ASA) use-dependent cut-offs can be used for outcome analysis in mixed populations of aspirin (ASA) users and non-users.

[0096] The most consequential finding of this study was the observation that adjusting raw urinary TXB2-M values for eGFR in addition to urine creatinine significantly improved the prognostic performance of the assay. Stable prostanoid metabolites, including TXB2-M, are filtered by the kidney and therefore their urine concentration is a function of plasma concentration, the rate of glomerular filtration and the amount of water reabsorption in the proximal tubule and descending loop of Henle. To account for variations in urine volume and concentration, raw TXB2-M concentrations have historically been normalized to the concentration of urine creatinine, which exhibits marked intra-individual variation throughout the day. The rationale for additionally adjusting the raw urinary TXB2-M concentration to eGFR arose from the observation in two different populations that urinary TXB2-M and renal function were independently and directly associated, irrespective of aspirin (ASA) use (Rade JJ, Barton BA, Vasan RS, Kronsberg SS, Xanthakis V, Keaney JF, Jr., et al. Association of thromboxane generation with survival in aspirin users and nonusers.J Am Coll Cardiol 2022;80:233-50; Kakouros N, Nazarian SM, Stadler PB, Kickler TS, Rade JJ. Risk factors for non-platelet thromboxane generation after coronary artery bypass graft surgery. Journal of the American Heart Association 2016;5:e002615). While daily eGFR does not vary as dramatically as urine concentration in a given individual, it can change substantially over time due to disease activity or medication use. Not only did the use of optimized urinary TXEL-M / eGFR cut-points (16.6 and 62.1 FPU for aspirin (ASA) users and non-users, respectively) result in better all-cause mortality risk discrimination, their superiority for assessing cardivascular / stroke mortality risk was also demonstrated.

[0097] The utility of assessing systemic TXA2 generation by measuring TXB2- M / eGFR rather than TXB2-M is likely greatest in heterogenous populations where renal function is impaired or fluctuates. It was previously demonstrated that systemic TXA2 generation is proportional to the degree of clinical heart failure and impairment of cardiac performance (Hariri E, Kakouros N, Bunsick DA, Russell SD, Mudd JO, Laws K, et al. Nonplatelet thromboxane generation is associated with impaired cardiovascular performance and mortality in heart failure. Am J Physiol Heart Circ Physiol 2022;323:H248-h55). Decompensated heart failure is associated with impaired renal perfusion and diminished eGFR that would reduce TXB2-M filtration and thus bias the association between systemic TXA2 generation and urinary TXB2-M (Zannad F, Rossignol P. Cardiorenal syndrome revisited. Circulation 2018;138:929-44). There is mechanistic evidence implicating nonplatelet TXA2 generation in the pathobiology of heart failure, and it is plausible that inhibiting it could improve cardiac function and outcome (West JD, Voss BM, Pavliv L, de Caestecker M, Hemnes AR, Carrier EJ. Antagonism of the thromboxane-prostanoid receptor is cardioprotective against right ventricular pressure overload. Pulm Circ 2016;6:211-23; West JD, Galindo CL, Kim K, Shin JJ, Atkinson JB, Macias-Perez I, et al. Antagonism of the thromboxane-prostanoid receptor as a potential therapy for cardiomyopathy of muscular dystrophy. J Am Heart Assoc 2019;8:e011902; Touchberry CD, Silswal N, Tchikrizov V, Elmore CJ, Srinivas S, Akthar AS, et al. Cardiac thromboxane a2 receptor activation does not directly induce cardiomyocyte hypertrophy but does cause cell death that is prevented with gentamicin and 2-apb. BMC Pharmacol Toxicol 2014;15:73; Wacker MJ, Kosloski LM, Gilbert WJ, Touchberry CD, Moore DS, Kelly JK, et al. Inhibition of thromboxane a2- induced arrhythmias and intracellular calcium changes in cardiac myocytes by blockade of the inositol trisphosphate pathway. J Pharmacol Exp Ther 2009;331 :917-24). Adjusting urinary TXB2-M for eGFR would therefore be important in longitudinal studies of systemicTX Az generation in heart failure and other populations with high inter- and intra-individual variations in renal function.

[0098] As the association of systemic TXA2 generation with mortality risk is independent of many traditional cardiovascular disease (CVD) risk factors, the findings here raise the possibility that measurement and inclusion of TXEF-M / eGFR could improve risk assessment if incorporated into cardiovascular risk models and calculators. This hypothesis is eminently testable using several existing large longitudinal population datasets. It was previously found that measurement of systemic TXA2 generation in individuals without cardiovascular disease (CVD) could identify those who might benefit from or be harmed by the use of aspirin (ASA) for primary prevention of cardiovascular disease (CVD) (Rade JJ, Barton BA, Vasan RS, Kronsberg SS, Xanthakis V, Keaney JF, Jr., et al. Association of thromboxane generation with survival in aspirin users and nonusers. J Am Coll Cardiol 2022;80:233-50). The identification of aspirin (ASA) use-specific optimized TXB2-M / eGFR cut-points would also facilitate the design of a prospective trial addressing this intriguing question.

[0099] While renal function can be characterized by multiple different parameters, creatinine-based estimation of GFR is the most useful clinically it is noted that it can also be estimated by multiple formulae. The Chronic Kidney Disease Epidemiologic Collaboration 2021 (CKD-EPI 2021) race-independent formula for estimating eGFR is the method currently endorsed by the National Kidney Foundation and has become clinical laboratory standard (Delgado C, Baweja M, Crews DC, Eneanya ND, Gadegbeku CA, Inker LA, et al. A unifying approach for gfr estimation: Recommendations of the nkf-asn task force on reassessing the inclusion of race in diagnosing kidney disease. Am J Kidney Dis 2022;79:268-88. el). Optimized cut-points of TXB2-M / eGFR were also determined using eGFR based on the race-dependent Modification of Diet in Renal Disease (MDRD) formula and found that they differed slightly from those based on the Chronic Kidney Disease Epidemiologic Collaboration 2021 (CKD-EPI 2021) formula, though the associations of the cut-points with outcome were essentially identical (Tables 9-11). While this reaffirms the concept that adjusting for renal function improves the prognostic power of measurements of urinary TXA2 metabolites, it introduces a note of caution that specific cut-points will be dependent on which metabolite is measured and how eGFR is calculated.

[0100] The Framingham Heart Study (FHS) Offspring enrolled individuals predominantly of northern European ancestry. Even with inclusion of the more diverse Omni Cohort, non- white participants accounted for <10% of study population. As race has beendemonstrated to be an independent risk factor for systemic TXA2 generation (akouros N, Nazarian SM, Stadler PB, Kickler TS, Rade JJ. Risk factors for non-platelet thromboxane generation after coronary artery bypass graft surgery. Journal of the American Heart Association 2016;5:e002615), it is possible that the optimized TXB2-M and TXB2-M / eGFR cut-points might differ among racial and ethnic groups. Future studies utilizing more racially diverse populations will be able to explore this possibility.

[0101] The present analysis could not account for changes in aspirin (ASA) use over time in the Framingham Heart Study (FHS) population and it is possible that such changes could affect outcome risk but would not be expected to alter the comparative relationships between outcome risks using the different cut-points.

[0102] The urine samples obtained from Framingham Heart Study (FHS) participants used in this study had been in long-term frozen storage, and it is possible that TXB2-M may have degraded over time. The stability of 1 l-dehydoTXB2 in urine samples frozen at -80°C for up to 9 years was previously studied (Olson MT, Kickler TS, Lawson JA, McLean RC, Jani J, FitzGerald GA, Rade JJ. Effect of assay specificity on the association of urine 11- dehydro thromboxane b2 determination with cardiovascular risk. J Thromb Haemost 2012;10:2462-9). Several points argue against a significant degradation of TXB2-M caused by long-term storage and an effect on the analyses in this study. First, analyte degradation would be expected to reduce the association of TXB2-M with survival outcomes, which was nonetheless very robust. Second, analyte degradation would not be expected to alter the comparative relationship between TXB2-M and TXB2-M / eGFR to outcome. Finally, results from the primary study population were externally validated in a population where urine samples were frozen for only a few months prior to batch analysis (Hariri E, Kakouros N, Bunsick DA, Russell SD, Mudd JO, Laws K, et al. Nonplatelet thromboxane generation is associated with impaired cardiovascular performance and mortality in heart failure. Am J Physiol Heart Circ Physiol 2022;323:H248-h55).Table 9. Optimized urinary TXB2-M / eGFR cut-points for all-cause mortality risk in Framingham Heart Study participants stratified by aspirin (ASA) use.*N=1352 and 1661 for aspirin (ASA) users and non-users, respectively.Abbreviations for Table 9: ASA, aspirin; CI, confidence interval; CKD-EPI, Chronic Kidney Disease Epidemiologic Collaboration; eGFR, estimated glomerular filtration rate; FPU, filtered prostanoid unit (1 FPU =pg TXBz-M min / urine creatinine-mL- 1.73m2); HR, hazard ratio; MDRD, Modification of Diet in Renal Disease; N / A, not applicable; TXB2-M, thromboxane B2 metabolites.Table 10. Relative differences in all-cause mortality risk and survival times in a cohort of3013 Framingham Heart Study participants using different aspirin (ASA) use-dependent urinary TXB2-M cut-points.*P <0.0001 versus AIC for 4thquartile cut-point model.#P <0.0001 versus AIC for optimized TXB2-M and 4thquartile cut-point models. fTo convert pg / mg creatinine to pg / pmol creatinine divide by 8.842.Abbreviations for Table 10: AIC, Akaike Information Criterion; ASA, aspirin; CI, confidence interval; CKD-EPI, Chronic Kidney Disease Epidemiologic Collaboration; eGFR, estimated glomerular filtration rate; FPU, filtered prostanoid unit ( 1 FPU =pg-min / creatinine-mL- 1 .73m2); HR, hazard ratio; MDRD, Modification of Diet in Renal Disease; RMST, restricted mean survival time; TXB2-M, thromboxane B 2 metabolites.Table 11. Relative cardiovascular and stroke mortality risk and survival times in a cohort of 3013 Framingham Heart Study participants using different aspirin (ASA) use-dependent urinary TXB2-M cut-points.*P =0.03 versus AIC for 4thquartile cut-point model.#P <0.0001 versus AIC for optimized TXB2-M and 4thquartile cut-point models.Abbreviations for Table 11: AIC, Akaike Information Criterion; ASA, aspirin; CI, confidence interval; CKD-EPI, Chronic Kidney Disease Epidemiologic Collaboration; eGFR, estimated glomerular filtration rate; FPU, filtered prostanoid unit ( 1 FPU=pg-mirFcreatinine-mL-1.73m2); HR, hazard ratio; MDRD, Modification of Diet in Renal Disease; RMST, restricted mean survival time; TXB2-M, thromboxane B 2 metabolites.

[0103] Thus, optimized urinary TXB2-M and TXB2-M / eGFR cut-points for aspirin (ASA) users and non-users were determined in a large unselective population, externally validated in aspirin (ASA) users with heart failure (HF), and it was demonstrated that adjusting for renal function in addition to urine concentration improves the prognostic power of this assay.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting thromboxane B2 metabolites (TXB2-M) in a urine sample, the method comprising, consisting essentially or, or consisting of: determining the amount of thromboxane B2 metabolites (TXB2-M) in the urine sample, and adjusting or normalizing the amount of thromboxane B2 metabolites (TXB2-M) to creatinine (e.g., urine creatinine or creatinine level) in the urine sample (e.g., picogram (pg) of thromboxane B2 metabolites (TXB2-M) / milligram (mg) of creatinine).

2. The method of claim 1 , wherein at least one of:(a) the amount of thromboxane B2 metabolites (TXB2-M) is determined by enzyme- linked immunosorbent assay (ELISA), mass spectrometry, western blot, nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, High / Ultra Performance Liquid Chromatography (H / UPLC), Liquid Chromatography Mass Spectrometer (LC-MS), Gas Chromatography Mass Spectrometer (GC-MS) technology, or a combination thereof;(b) the creatinine is determined by enzyme-linked immunosorbent assay (ELISA), mass spectrometry, western blot, nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, High / Ultra Performance Liquid Chromatography (H / UPLC), Liquid Chromatography Mass Spectrometer (LC- MS), Gas Chromatography Mass Spectrometer (GC-MS) technology, or a combination thereof; or(c) a combination thereof.

3. The method of claim 1 or 2, wherein the amount of thromboxane B2 metabolites (TXB2-M) adjusted or normalized to creatinine (e.g., pg / mg) in the urine sample is adjusted or normalized to renal function.

4. The method of claim 3, wherein the renal function includes or is estimated glomerular filtration rate (eGFR) (e.g., TXB2-M / eGFR, such as pg-min / creatinine-mL- 1.73m2or a filtered prostanoid unit (FPU)).

5. The method of claim 4, wherein the estimated glomerular filtration rate (eGFR) is determined by Chronic Kidney Disease Epidemiology Collaboration 2021 (CKD- EPI 2021) formula based on serum creatinine.

6. A method of examining or determining mortality risk in a subject, the method comprising, consisting essentially of, or consisting of, performing the method of claim 1 or 2,and determining or assigning the mortality risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) in the urine sample.

7. The method of claim 6, wherein a subject with a thromboxane B2 metabolites (TXB2-M) level of at least about 1291 pg / mg creatinine (e.g., about 1291 or greater pg / mg creatinine) for aspirin (ASA) users or at least about 5609 pg / mg creatinine (e.g., about 5609 or greater pg / mg creatinine) for non-aspirin (non-ASA) users is at increased mortality risk.

8. The method of claim 7, wherein the subject is at increased mortality risk from a disease, disorder, or condition that includes or is at least one of cardiovascular disease, heart failure (e.g., heart failure with a preserved ejection fraction or heart failure with a reduced ejection fraction), stroke, or a combination thereof.

9. The method of claim 8, wherein the disease, disorder, or condition is heart failure, and the subject has (1) two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria and two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) minor criteria, wherein at least one of: each of the major criteria includes or is independently selected from: paroxysmal nocturnal dyspnea or orthopnea; distended neck veins (in other than the supine position); rales; increasing heart size by x-ray; acute pulmonary edema on chest x-ray; ventricular S3 gallop; increased venous pressure > 16 cm H2O; hepatojugular reflux; pulmonary edema, visceral congestion, and / or cardiomegaly shown on autopsy; weigh loss on heart failure treatment (e.g., 10 pounds / 5 days); and a combination thereof; each of the minor criteria includes or is independently selected from: bilaterial ankle edema, night cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion by x-ray, decrease in vital capacity by one-third from maximum record, tachycardia (e.g., > 120 beats per minute), pulmonary vascular engorgement on chest x-ray, and a combination thereof; or a combination thereof.

10. A method of examining or determining mortality risk in a subject, the method comprising, consisting essentially of, or consisting of, performing the method of any one of claims 3-5, and determining or assigning the mortality risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) in the urine sample.

11. The method of claim 10, wherein a subject with a TXB2-M / estimated glomerular filtration rate (eGFR) of about 16.6 pg-min / creatinine-mL- 1.73m2(e.g., about 16.6 or greater pg min / creatinine-mL- 1.73m2) for aspirin (ASA) users or about 62.1 pg-min / creatinine-mL- 1.73m2(e.g., about 62.1 or greater pg-min / creatinine-mL- 1.73m2) for non-aspirin (ASA) users is at increased mortality risk.

12. The method of claim 11 , wherein the subject is at increased mortality risk from a disease, disorder, or condition that includes or is at least one of cardiovascular disease, heart failure (e.g., heart failure with a preserved ejection fraction or heart failure with a reduced ejection fraction), stroke, or a combination thereof.

13. The method of claim 12, wherein the disease, disorder, or condition is heart failure, and the subject has (1) two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) major criteria and two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) minor criteria, wherein at least one of: each of the major criteria includes or is independently: paroxysmal nocturnal dyspnea or orthopnea; distended neck veins (in other than the supine position); rales; increasing heart size by x-ray; acute pulmonary edema on chest x-ray; ventricular S3 gallop; increased venous pressure > 16 cm H2O; hepatojugular reflux; pulmonary edema, visceral congestion, and / or cardiomegaly shown on autopsy; weigh loss on heart failure treatment (e.g., 10 pounds / 5 days); or a combination thereof; each of the minor criteria includes or is independently bilaterial ankle edema, night cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion by x-ray, decrease in vital capacity by one-third from maximum record, tachycardia (e.g., > 120 beats per minute), pulmonary vascular engorgement on chest x-ray, or a combination thereof; or a combination thereof.

14. A method of examining or determining heart failure risk in a subject, the method comprising, consisting essentially of, or consisting of, performing the method of any one of claims 3-5, and determining or assigning the heart failure risk in the subject based on the amount of thromboxane B2 metabolites (TXB2-M) adjusted / normalized to creatinine level (e.g., pg / mg) in the urine sample, adjusted or normalized to renal function.

15. The method of claim 14, wherein the subject does not have heart failure or has no history of heart failure.

16. The method of claim 14 or 15, wherein the subject has pre-heart failure (e.g., Stage A heart failure or Stage B heart failure according to The American College of Cardiology (ACC) and the American Heart Association (AHA) Stages of Heart Failure).

17. The method of any one of claims 14-16, wherein a subject with a TXB2- M / estimated glomerular filtration rate (eGFR) of about 16.6 pg-min / creatinine-mL- 1.73m2(e.g., about 16.6 or greater pg-min / creatinine mL- 1.73m2) for aspirin (ASA) users or about 62.1 pg-min / creatinine-mL- 1.73m2(e.g., about 62.1 or greater pg-min / creatinine-mL- 1.73m2) for non-aspirin (ASA) users is at increased risk of developing heart failure.

18. The method of any one of claims 8, 9, or 12-17, further comprising treating the subject for the disease, disorder, or condition.

19. The method of claim 18, wherein the disease, disorder, or condition is cardiovascular disease and the treatment includes or is at least one of a cholesterol lowering medication (e.g., statin, atorvastatin, simvastatin, rosuvastatin, pravastatin, fluvastatin, or a combination thereof), antiplatelet or blood thinning agent (e.g., aspirin, clopidogrel, rivaroxaban, ticagrelor, prasugrel, or a combination thereof), beta blocker (e.g., atenolol, bisoprolol, carvedilol, metoprolol, nebivolol, or a combination thereof), nitrate (e.g., hydralazine with nitrate), angiotensin-converting enzyme (ACE) inhibitor (e.g., ramipril, lisinopril, or a combination thereof), angiotensin-2 receptor blocker (such as candesartan, losartan, telmisartan, valsartan, or a combination thereof), calcium channel blocker (e.g., amlodipine, verapamil, diltiazem, or a combination thereof), diuretic (such as furosemide / frusemide, bumetanide, or a combination thereof), echocardiogram, angiogram, computed tomography (CT) scan, coronary angioplasty, (e.g., percutaneous coronary intervention (PCI), percutaneous transluminal coronary angioplasty (PTCA), or balloon angioplasty), coronary artery bypass grafting (CABG), heart transplant, omega-3 fatty acids, thromboxane- A synthase inhibitor (e.g., ridogrel, dazoxiben, imidazole, ozagrel, or a combination thereof), a thromboxane receptor antagonist (e.g., ridogrel, seratrodast, momiflumate, or a combination thereof), or a combination thereof.

20. The method of claim 18, wherein the disease, disorder, or condition is heart failure (e.g., heart failure with a preserved ejection fraction or heart failure with a reduced ejection fraction) and the treatment includes or is at least one of heart failure medication, angiogram (e.g., percutaneous coronary intervention (PCI), percutaneous transluminal coronary angioplasty (PTCA), or balloon angioplasty), pacemaker, cardiac resynchronization therapy (CRT) device, implantable cardioverter defibrillator (ICD), a cardiac resynchronization therapy defibrillator (CRT-D), or a combination thereof.

21. The method of claim 20, wherein the heart failure medication includes or is at least one of an antiplatelet or blood thinning agent (e.g., aspirin, clopidogrel, rivaroxaban, ticagrelor, prasugrel, or a combination thereof), ace inhibitor (e.g., ramipril, captopril, enalapril, lisinopril, perindopril, or a combination thereof), angiotensin-2 receptor blocker (e.g., candesartan, losartan, telmisartan, valsartan, or a combination thereof), beta blocker (e.g., atenolol, bisoprolol, carvedilol, metoprolol, nebivolol, or a combination thereof), mineralocorticoid receptor antagonist (e.g., spironolactone, eplerenone, or a combination thereof), diuretic (e.g., furosemide / frusemide, bumetanide, or a combination thereof),ivabradine, sacubitril valsartan, nitrate (e.g., hydralazine with nitrate), digoxin, SGLT2 inhibitor (e.g., empagliflozin, dapagliflozin, or a combination thereof), or combination thereof), thromboxane-A synthase inhibitor (e.g., ridogrel, dazoxiben, imidazole, ozagrel, or a combination thereof), a thromboxane receptor antagonist (e.g., ridogrel, seratrodast, momiflumate, or a combination thereof), glucagon-like peptide 1 (GLP-1) receptor agonist (e.g., dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, tirzepatide, polyethylene glycol loxenatide, or a combination thereof), or a combination thereof.

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