WO2016113719A1

WO2016113719A1 - Use of 1,25-dihydroxyvitamin d values in ratio with pth as a prognostic biomarker

Info

Publication number: WO2016113719A1
Application number: PCT/IB2016/050229
Authority: WO
Inventors: Damien GRUSON; Michel Rousseau; Claudia Zierold; Fabrizio Bonelli; Frank BLOCKI
Original assignee: Universite Catholique de Louvain UCL; Diasorin SpA
Current assignee: Universite Catholique de Louvain UCL; Diasorin SpA
Priority date: 2015-01-18
Filing date: 2016-01-18
Publication date: 2016-07-21
Anticipated expiration: 2017-07-18
Also published as: CA2973653A1; AU2016207734A1; EP3245515A1; WO2016113720A1

Abstract

The present invention relates to a method for predicting or stratifying the risk of morbidity and/or mortality in a chronic heart failure patient. Levels of 1,25-dihydroxyvitamin D (1,25(OH) ₂D) are measured in a biological sample and taken together with parathyroid hormone (PTH) levels to provide a ratio indicative of risk of chronic heart failure morbidity and/or mortality.

Description

Use of 1,25-dihvdroxyvitamin D values in ratio with PTH as a prognostic biomarker

FIELD OF THE INVENTION

The present invention relates to a method for predicting or stratifying the risk of morbidity and/or mortality in a chronic heart failure patient. Levels of 1,25-dihydroxyvitamin D (l,25(OH)₂D) are measured in a biological sample and taken together with parathyroid hormone (PTH) levels to provide a ratio indicative of risk of chronic heart failure morbidity and/or mortality.

BACKGROUND OF THE INVENTION

Cardiovascular diseases remain a leading cause of death around the world. Among cardiovascular diseases, heart failure (HF) represents a major health concern because of increasing prevalence worldwide, and major human, societal and economic impacts. The diagnostic assessment of patients with heart failure requires appropriate tools capable of providing predictive information on the apportioning of risk of death and/or morbidity, such as hospital readmission, transplantation and/or the use of ventricular assist device. Identifying heart failure patients likely to suffer poor outcomes is therefore essential for the appropriate management of these patients. On the other hand, as pointed out in Meijers WC et al, since in the recent years the number of survivors requiring rehospitalization owing to HF after an initial admission have risen steadily, the ability to identify a population of HF patients at low risk of early revisits and mortality would be beneficial (Meijers WC et al., Eur J Heart Fail. 2015; 17(12): 1271-82). Such a low risk indicator allows for early and safe discharge of a selected group of patients, at the same time allowing for more aggressive therapy in the remaining patient population.

The need for biomarkers for the prognosis of HF is well established, and different biomarkers from several pathophysiological pathways have been evaluated for this clinical indication. The diagnostic role of natriuretic peptides (NPs) in both acute and chronic HF is well established. In chronic HF, measurements of blood levels of either brain natriuretic peptide (BNP) or N-terminal-pro-BNP (NT-proBNP) provide independent information regarding the risk for disease progression across a wide spectrum of adverse outcomes. However, cross- reactivity between active and nonactive NP components may lead to the inappropriate interpretation of the NP that has been assayed, thereby affecting the diagnostic power of NP- based assays. As additional marker to the prognostic value of the NPs in HF patients, galectin- 3 has recently emerged as biomarker of myocardial fibrosis that is predictive of hospitalization and death. WO2005040817A1 discloses a method for the identification of patients who are at particular risk of complications of heart failure, which is based on the determination of the non-myocitical markers galectin-3.

Vitamin D deficiency and hyperparathyroidism are common in patients with HF. There is a growing body of evidence supporting the role of vitamin D and parathyroid hormone (PTH) in cardiac remodeling and worsening of HF (Gruson D, et al. Clin Chim Acta 2014; 433:290-6). Lack of reliable automated testing of 1, 25 -dihydroxy vitamin D (l,25(OH)₂D), the biologically active metabolite of vitamin D, has in the past limited evaluation of the prognostic value of this measurement

PTH may also be part of a vicious and deleterious cycle for cardiovascular function together with aldosterone and fibroblast growth factor 23 (FGF-23). Markedly elevated levels of FGF- 23 and PTH were observed in patients with cardiovascular disorders and HF, and were related to adverse cardiovascular events.

Therefore, there is a need to identify biomarkers for predicting or stratifying the risk of morbidity and/or mortality in a patient suffering from chronic heart failure.

SUMMARY OF THE INVENTION

The present invention provides a method for predicting or stratifying the risk of morbidity and/or mortality in a chronic heart failure patient using the level of l,25(OH)₂D in conjunction with the level of parathyroid hormone(PTH) to determine the ratio of l,25(OH)₂D and PTH. The ratio value allows for risk prediction or stratification of morbidity and/or mortality in a chronic heart failure patient.

The term "PTH" as used in the present description preferably refers to parathyroid hormone 1- 84 (PTH 1-84), which is the biologically active hormone produced by the parathyroid glands and secreted into the systemic circulation.

Therefore, the invention provides a method for predicting or stratifying the risk of morbidity and/or mortality in a chronic heart failure patient, comprising: (a) detecting and quantifying l,25(OH)₂D in a sample from the patient; (b) detecting and quantifying PTH in a sample from the patient; and (c) calculating the l,25(OH)₂D to PTH ratio, wherein when the ratio is above a predetermined threshold, the patient is predicted or stratified not to have an increased risk of chronic heart failure morbidity and/or mortality, and when the ratio is below a predetermined threshold, the patient is predicted or stratified to have an increased risk of chronic heart failure morbidity and/or mortality.

The measurement of the ratio of l,25(OH)₂D to PTH in chronic heart failure patients offers several advantages such as the improvement of the area under the receiver operating curve, translating the added clinical value, the integration of more than one biomarker physiopathologicaly interrelated and the modulation of two different hormones to increase the value of the ratio.

In various embodiments, l,25(OH)₂D and/or PTH are determined from blood, plasma, serum, saliva, lymph, urine or feces samples from the patient. In these and other embodiments, l,25(OH)₂D and/or PTH are determined using an immunoassay. In particular embodiments, the immunoassay is a chemiluminescent assay.

In one embodiment, when the ratio of l,25(OH)2D to PTH is below the predetermined threshold, the chronic heart failure patient is stratified as having a high risk of chronic heart failure morbidity and/or mortality.

In another embodiment, when the l,25(OH)₂D to PTH ratio is above the predetermined threshold, the chronic heart failure patient is stratified as having a low risk of chronic heart failure morbidity and/or mortality.

Preferably, the predetermined threshold in these embodiments is comprised within the range of from 0.9 to 1.4, more preferably the predetermined threshold is 1.06.

In still yet another embodiment, l,25(OH)₂D is determined using an immunoassay which comprises (i) contacting the l,25(OH)₂D in the sample from the patient with a receptor protein comprising the Ligand Binding Domain of Vitamin D Receptor (VDR-LBD), thereby obtaining a first complex; (ii) contacting said first complex with a capture moiety that specifically binds to a conformational epitope on said first complex, but does not bind to either l,25(OH)₂D or VDR-LBD that is not bound in said first complex, thereby obtaining a second complex; and (iii) detecting and quantitating said second complex as an indication of the amount of l,25(OH)₂D in the sample.

In a preferred embodiment, the capture moiety is a monoclonal antibody. Preferably, the capture moiety is immobilized on a solid support.

In another preferred embodiment, the immunoassay of the claimed method is a sandwich immunoassay.

In a more preferred embodiment, step (iii) of detecting and quantitating said second complex is carried out by means of a labeled anti- VDR-LBD detector antibody.

Further, while the present l,25(OH)₂D data was obtained using a new immunoassay that provides rapid, sensitive and reproducible data using significantly smaller volumes of samples than other available assays, those of skill in the art will appreciate that any method of collecting reliable values for l,25(OH)₂D and PTH is contemplated. For example, such methods may include GC-MS, LC-MS/MS and the like.

These and other features and advantages of the present invention will be set forth or will become more fully apparent in the description that follows and in the appended claims. The features and advantages may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. Furthermore, the features and advantages of the invention may be learned by the practice of the invention or will be apparent from the description, as set forth hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

Various exemplary embodiments of the compositions and methods according to the invention will be described in detail, with reference to the following figures wherein:

Figure 1. Receiver operating characteristic (ROC) analysis were performed for cardiovascular death at the end of the follow-up for 25(OH) vitamin D, FGF-23, l,25(OH)₂ vitamin D and its ratio to PTH.

Figure 2. Kaplan-Meier survival curve stratified by [l,25(OH)₂D]/[PTH] ratio.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", "characterized by" and "having" can be used interchangeably. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, instruments, statistical analyses and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Abbreviations used throughout this text are as follows: HF, Heart Failure; NYHA, New York Heart Association; PPV, Positive Predictive Value; NPV, Negative Predictive Value; EF, left ventricular ejection fraction; GFR, glomerular filtration rate; CV, cardiovascular; BNP, B-type natriuretic peptide; NT-proBNP, N-terminal B-type natriuretic peptide; Gal-3, Galectin-3; l,25(OH)₂D, 1, 25 -dihydroxy vitamin D; PTH, parathyroid hormone; ROC, receiver operating characteristics; AUC, area under the curve; FGF-23, fibroblast growth factor 23, and CgA, chromogranin A.

As used herein the term "morbidity" means the status of chronic heart failure disease, particularly as evaluated based on re -hospitalization, transplantation and/or the use of ventricular assist device.

25 -Hydroxy vitamin D (25(OH)D) (calcidiol) has been previously shown to be associated with cardiovascular events, however, under conditions of vitamin D (cholecalciferol) deficiency, this biomarker may not be as predictive as its biologically active metabolite 1,25- dihydroxyvitamin D (l,25(OH)₂D) or calcitriol. l,25(OH)₂D, PTH, and FGF-23 control calcium and phosphate homeostasis (Scialla JJ, Wolf M. Nat Rev Nephrol 2014; 10(5):268- 278). The potential value of l,25(OH)₂D testing as a significant predictor of cardiovascular events in HF patients was pursued in a model HF population.

The present study was designed using a new, fully-automated l,25(OH)₂D assay with improved analytical performance, sensitivity, and reliability. The inventors tested the hypothesis that levels of l,25(OH)₂D and its ratio to PTH are biomarkers that predict worsening of the disease, morbidity or cardiovascular death in HF patients.

Prior determinations for Vitamin D sufficiency have relied upon determining levels of circulating 25-Hydroxyvitamin D (25(OH)D)(calcidiol), which is produced in the liver by hydroxylation of vitamin D (cholecalciferol) but which is biologically inactive. 25(OH)D is used for such determinations as bone weakness, bone malformation, or abnormal metabolism of calcium (reflected by abnormal calcium, phosphorus, PTH) occurring as a result of a deficiency or excess of vitamin D. However, circulating 25(OH)D is transported to the kidneys where it is converted to its active form l,25(OH)₂D (calcitriol). l,25(OH)₂D acts on the gastrointestinal tract to promote the absorption of dietary calcium, acts upon the kidney to increase renal tubular reabsorption of calcium, and on the bone to mobilize calcium. l,25(OH)₂D circulates in the blood bound to the vitamin D binding protein, and enters target cells where the l,25(OH)₂D is made available to bind to the vitamin D receptor (VDR). This ligand/receptor complex readily translocates across the nuclear membrane to act as a transcription factor. l,25(OH)₂D is now known to have a broader spectrum of action, and has been associated with increased risks for various chronic conditions including infectious and autoimmune, diabetes, cancer, cardiovascular ailments, hypertension, obesity and overweight and complications during pregnancy. Therefore, the inventors hypothesized that l,25(OH)₂D levels may be more indicative of homeostatic health and aberrations therefrom as manifested in the heart, intestine, immune, bone, neuronal degeneration, cancer and diabetes. While values for serum l,25(OH)₂D are not generally taken, it is considered that normal circulating levels of l,25(OH)₂D in the U.S. are in the range of 19.9-79.3 pg/ml with a median of about 49.6 pg/ml.

Since the binding of l,25(OH)₂D to VDR-LBD is known to induce a conformational change in VDR-LBD, immunoassay methods for detecting total l,25(OH)₂D may involve the use of a conformation-specific capture moiety, such as an antibody, capable of specifically recognizing and binding to VDR-LBD bound to l,25(OH)₂D, in order to selectively discriminate the VDR- LBD/l,25(OH)₂D complex from either l,25(OH)₂D or unbound VDR-LBD, as described in WO2014114780. Preferably, the capture moiety of the method of the invention is a monoclonal antibody.

Furthermore, in such detection methods, the detection of the captured VDR-LBD/l,25(OH)₂D complex may be accomplished through a detectable signal, which is generated directly, for example, by employing a labeled receptor protein or indirectly, for example, via a labeled detector molecule which is capable of specifically binding the VDR-LBD/l,25(OH)₂D complex captured by the capture moiety. Typically, the detector molecule is an antibody directed to an epitope on the VDR-LBD/l,25(OH)₂D complex which is different from the epitope recognized by the capture moiety.

According to a preferred embodiment, the l,25(OH)₂D immunoassay of the method of the invention is a sandwich immunoassay, more preferably a chemiluminescence immunoassay. Depending on the format of the immunoassay, the capture antibody may be immobilized on a solid support. Non limiting examples of suitable solid supports are the wells of a microtitre plate, the surface of a microparticle such as a latex, polystyrene, silica, chelating sepharose or magnetic beads, membranes, strips or chips.

PTH is secreted by the parathyroid glands and acts to increase the concentration of calcium in the blood by binding to the parathyroid receptor, having high levels in bone and kidney) and parathyroid hormone 2 receptor (CNS, pancreas, testis and placenta). Further, PTH increases the activity of the 1-a-hydroxylase which converts 25-hydroxyvitamin D to l,25(OH)₂D in the kidney to support endocrine functions and 1-a-hydroxylase is also expressed in various other tissues, whose cells may convert 25(OH)D for autocrine and paracrine functions. Normal values for PTH are considered to be 10 to 55 pg/ml. The median for the ratio of l,25(OH)₂D to PTH in normal individuals is approximately 2.7 (range of 1.2-9.1).

There is a growing body of evidence supporting the role of vitamin D and (PTH) in cardiac remodeling and worsening of HF. Furthermore, PTH may also be part of a vicious and deleterious cycle for cardiovascular function together with aldosterone and fibroblast growth factor 23 (FGF-23) (Tomaschitz A, et al. Cardiovasc Res 2012; 94(1): 10-19). Markedly elevated levels of FGF-23 and PTH were observed in patients with cardiovascular disorders and HF, and were related to adverse cardiovascular events.

The ratio of l,25(OH)₂D to PTH is identified herein as being a biomarker for predicting or stratifying the risk of morbidity and/or mortality in a chronic heart failure patient.

Various exemplary embodiments of devices and compounds as generally described above and methods according to this invention, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention in any fashion.

EXAMPLE 1 : l,25(OH)₂D:PTH Biomarker of HF

MATERIAL AND METHODS

Study population: The cohort studied, recruited at the Cliniques Universitaires Saint-Luc, an academic hospital of Brussels, Belgium, consisted of outpatients with primarily chronic HF with reduced left ventricular ejection fraction. Each patient gave informed consent and the local institutional review board approved the protocol. The primary inclusion criterion was a clinical diagnosis of HF with a left ventricular ejection fraction (EF) <35% determined by contrast or isotopic ventriculography. Participants were excluded if they had a non-cardiac condition resulting in an expected mortality of <6 months as judged by the treating physician, or if they were unable or unwilling to provide informed consent. At the time of study entry, detailed clinical data was obtained using a standardized questionnaire administered to the patient by the treating physician, and further verified by medical records. Venous blood samples were obtained at enrollment, processed, and stored at -80°C until time of assay.

Clinical Endpoints: The primary outcome measured in this study was defined as cardiovascular (CV) mortality. Follow-up events, including CV mortality and cardiac transplantation, were prospectively ascertained every 6 months via direct patient contact, and verified through death certificates, medical records, and contact with patients' family members by dedicated research personnel.

Laboratory measurements: Routine laboratory measurements and blood samples for biomarker analysis were obtained at hospital admission. EDTA plasma and serum were separated and stored at -80°C until further analysis. Levels of l,25(OH)₂D were determined at baseline with a fully automated and sensitive immunoassay that uses a specific recombinant fusion protein for the capture of l,25(OH)₂D (DiaSorin, Saluggia, Italy, #310980). The measurement range of this assay is between 5 and 200 pg/ml with the limit of detection being 0.70 pg/ml and the limit of quantitation being 5.0 pg/ml. The reference interval determined in healthy volunteers ranged between 19.9-79.3 pg/ml with a median of about 49.6 pg/ml.

PTH levels were determined using a sensitive immune assay for the determination of PTH in blood, serum or plasma (Liaison 1-84 PTH, DiaSorin, Saluggia, Italy, #310630) with a measurement range of between 4 and 1800 pg/ml, with the limit of detection being 1.7 pg/ml and the limit of quantitation being 4 pg/ml. The reference interval determined for healthy 25(OH)D sufficient, volunteers ranged from 6.5-36.8 with a median of 15.3 pg/ml.

Plasma Q-FGF23 concentrations were determined at entry into the study with a second- generation C-terminal human enzyme-linked immunosorbent assay (Immutopics, San Clemente, CA, USA), and circulating concentrations expressed as RU/ml. Levels of 25(OH)D, PTH, B-type natriuretic peptide (BNP), N-terminal proBNP (NT-proBNP) and Galectin-3 (Gal-3) were also determined. Glomerular filtration rate (eGFR) was estimated by the Modification of Diet in Renal Disease formula.

Statistical analysis: Continuous variables were expressed as medians with their interquartile values and range. Associations between l,25(OH)₂D and relevant clinical variables were tested using ANOVA for symmetric continuous, Kruskal-Wallis tests for non-symmetric continuous, and χ2 tests for categorical variables. The non-parametric Spearman rank correlation coefficients were used to assess the relationships between biomarkers. Additionally, independent determinants of baseline l,25(OH)₂D levels were assessed using multivariable linear regression methods with log transformed levels of l,25(OH)₂D as the dependent variable.

The discrimination power between biomarkers was assessed by analysis of their area under the receiver operating characteristic (ROC) curve. In a first step, the effects of survival age, EF and the various biomarkers, expressed as categorical variables on the basis of their medians, was assessed by the univariate COX proportional hazard analysis. In a second step, the influence of all variables was assessed in a stepwise backward multivariate COX analysis model. For the biomarkers remaining in the multivariate model, an ROC analysis was performed to determine the value of the parameter providing the maximal difference between sensitivity and (1 -specificity). The survival curve of patients below and above this value was established and compared by the Log-Rank test. P-values < 0.05 were considered significant. Statistical analysis was performed using MedCalc® software.

RESULTS

Study Population - Baseline characteristics: The distributions of baseline characteristics and laboratory values across the entire cohort and according to 25(OH)D and l,25(OH)₂D levels are displayed in Table 1. The cohort included 170 chronic HF patients (mean age 67±14 years; females n=36; males n=134; NYHA II-IV; etiology: ischemic n=119, dilated cardiomyopathy n=51; mean LVEF 23+7%).

In the present study, patients were classified according to the The New York Heart Association (NYHA) Functional Classification. Table 1

25(OH)D less 25(OH)D greater than P-value l,25(OH) l,25(OH)₂D greater P-value

Entire cohort ^{tnan me}dian median ₂D less than median

Characteristics than

median

69 66 70 0.559 70 67 0.116

Age (years)

[21 - 89] [33 - 89] [21 - 87] [33 - 89] [21 - 86] Sex (M/F) 134/36 64/21 70/15 0.808 67/18 67/18 0.785

Dilated 0.016 26 34 0.016 cardiomyopathy

(%)

Ischemic 74 66 0.022 74 66 0.022 cardiomyopathy

(%)

24 23 24 0.119 23 23 0.970

EF (%) [8 - 35] [8 - 35] [9 - 35] [9 - 35] [8 - 79 79 78 0.893 79 78 0.795

Heart Rate [46 - 135] [50- 135] [46 - 124] [51 - 135] [46 - 130]

Diabetes (%) 31 26 0.095 38 20 0.038

Hypertension (%) 55 49 61 0.031 45 55 0.047

Previous 32 27 34 0.045 38 41 0.112 admission to

hospital (%)

Smoker C/F/N 16/25/59 21/20/59 12/31/57 0.108 15/27/58 18/24/58 0.467 (%)

Treatment

ACE inhibitors 79 70 85 0.037 76 77 0.324 (%)

β-blockers (%) 83 83 81 0.134 86 80 0.112

Diuretics (%) 72 76 74 0.250 85 64 <0.001

Aldosterone 62 60 75 0.027 73 61 0.030 antagonists (%)

Angiotensin II 20 25 17 0.017 26 18 0.023 receptor blockers

(%)

Vitamin D (%) 3 2 4 0.346 0.076

Anticoagulant 38 40 35 0.332 41 35 0.276 (%)

Antiplatelet (%) 67 73 60 0.030 68 66 0.649

Antidiabetic drug 28 30 25 0.112 36 20 0.024 (%)

eGFR 56.1 55.6 58.5 0.958 51.6 61.6 0.001

(mL/min/l .73m2) [9.4 - 144.6] [9.4 - 144.6] [11.0 - 114.7] [9.4 [22.2 - 144.6]

107.8]

Calcium, total 8.9 8.9 8.9 0.320 8.8 9.0 0.007

(mg/dL) [6.2-10.8] [6.2-10.8] [7.8-10.4] [6.2 - [7.8 - 10.8]

10.4]

97 99 97 0.434 91 100 0.459

[16-220] [16-185] [32-220] [16-180] [32-220]

LDL-C (unit)

90 90 91 0.522 85 99 0.256

Triglycerides [14-345] [14-345] [28-282] [14-231] [39-345]

(unit)

12.3 8.5 18.8 < 0.001 10.9 15.0 0.024

25(OH)D [4.3-46.1] [4.3-12.1] [12.5-46.1] [4.3 - [5.0-46.1]

(ng/mL) 34.0]

25.4 22.5 28.4 0.025 17.4 35.8 <0.001 l,25(OH)₂D

[5.0-100] [5.0-74.1] [7.5-100] [5.0 - [25.6- 100]

(pg/mL)

25.2]

45 52 37 <0.001 44 45 0.024

PTH 1-84 [4-244] [12-244] [4-201] [8.7 - [4-201]

(pg/mL) 244]

455 756 308 <0.001 687 299 <0.001

[17-5017] [21-5017] [17-4408] [43 - [17-4408]

BNP (ng/L) 5017]

2157 3385 1571 <0.001 3276 929 <0.001

Nt-proBNP [66 - 33020] [66 - 33020] [95 - 21295] [71 - [66-29925]

(ng/L) 33020]

Most patients had levels of 25(OH)D below 30 ng/mL, and stratification by NYHA functional class did not disclose significant differences (p = 0.249) in values for 25(OH)D. In contrast, median serum levels of l,25(OH)₂D decreased significantly according to HF severity: 33.3 pg/mL in NYHA class II (n=60), 23.4 pg/mL in NYHA class III (n=94), and 14.0 pg/mL in NYHA class IV (n=16; p<0.001). The median l,25(OH)₂D level for all patients was 25.4 pg/mL (range: 5.0-100.0 pg/mL). The median for PTH levels for all patients was 38.0 pg/mL (range: 4.0-244 pg/mL). The median PTH levels by severity were as follows: NYHA class II 27.6 pg/mL, NYHA class III 41.4 pg/mL, and NYHA class IV 41.0 pg/mL.

The inventors examined the ratio of l,25(OH)₂D to PTH and found it to be significantly related to HF severity: NYHA class II ratio=1.14, NYHA class III ratio=0.47, and NYHA class IV ratio=0.38 (p<0.001). The l,25(OH)₂D to PTH ratios were higher in patients with l,25(OH)₂D levels above the median, while PTH was not different between the two groups.

Participants with l,25(OH)₂D levels below 35.4 pg/ mL were more likely to have higher circulating levels of BNP, NT-proBNP and Galectin-3. The l,25(OH)₂D to PTH ratio showed significant negative correlation with BNP, NT-proBNP FGF-23 and Gal-3, and 25(OH)D levels were correlated to BNP and NT-proBNP only. In multiple regression analysis, age, eGFR, PTH, and Galectin-3 were independent determinants of baseline l,25(OH)₂D.

L25(OH)₉D level and L25(OH)₉D to PTH ratio outcomes in chronic HF

Over a median follow-up time of 4.1 years (minimum and maximum follow-up times were 7 days and 7.4 years, respectively), 106 HF patients met the endpoint, i.e. 94 died and 12 underwent heart transplant. Biomarker concentrations as well as clinical variables between patients that developed the outcome and those who remained stable are presented in Table 2. Table 2

Variables Stable patients Patients with outcomes P-value

N = 64 N = 106

Age (years) 60.4 68.6 0.001

Sex (M/F) 47/17 87/19 0.153

Dilated cardiomyopathy (%) 42 22 0.046

Ischemic cardiomyopathy (%) 58 78 0.042

EF (%) 23.7 20.3 0.003

Heart Rate 79 79 0.897

Diabetes (%) 28.8 37.6 0.303

Hypertension (%) 55.3 62.4 0.358

Previous admission to hospital (%) 31.8 62.4 <0.001

Treatment

ACE inhibitors (%) 73 77 0.203 β-blockers (%) 63.0 77.8 0.048

Diuretics (%) 63.0 75.6 0.076

Aldosterone antagonists (%) 63.0 64.4 0.521

Angiotensin II receptor blockers (%) 20.7 24.0 0.655

Vitamin D (%) 1 3 0.487

Anticoagulant (%) 37.6 38.2 0.196

Antiplatelet (%) 67.1 62.4 0.633

Antidiabetic drug (%) 28.2 37.6 0.366 eGFR (mL/min/1.73m2) 61.7 49.2 0.002

Calcium, total (mg/dL) 8.9 8.8 0.955

BNP (ng/L) 227 666 < 0.001

NT-proBNP (ng/L) 1012 2992 < 0.001

Gal-3 (ng/mL) 16.3 19.5 0.001

CgA (UI/L) 26.6 47.2 < 0.001

FGF-23 (RU/mL) 149 413 < 0.001

PTH (l-84)(pg/mL) 28 41 0.001

25(OH)D (ng/mL) 13.1 12.4 0.488 l,25(OH)₂D (pg/mL) 31.7 20.2 < 0.001 l,25(OH)₂D / PTH(l-84) ratio 1.12 0.49 < 0.001

(l,25(OH)₂D)² / PTH(l-84) Square 35.38 9.91 < 0.001 ratio

COX proportional hazard (CPH) analysis revealed l,25(OH)₂D and its ratio to PTH to be strongly predictive of the outcome. In univariate COX survival analysis, l,25(OH)₂D and the ratio to PTH levels were significantly related to long-term cardiovascular death (p<0.01). The present analysis revealed that the coefficient of Log l,25(OH)₂D is roughly -2 times that of Log PTH, suggesting that the two might be summarized by a score function -2 Log l,25(OH)₂D + Log PTH, which is the negative Log of the ratio of the square of the l,25(OH)₂D assay to the PTH. This composite score marker is assessed in Table 3 as "Square ratio" whereas the l,25(OH)₂D to PTH ratio, without the doubling on the l,25(OH)₂D, is assessed under the label "Ratio". Table 3 shows that both the l,25(OH)₂D/PTH ratio and the [l,25(OH)₂D] /PTH ratio are highly competitive risk scores, being outperformed only by BNP and NT-proBNP, respectively.

Table 3

In ROC analysis, the area under the curve (AUC), criteria defined as cardiovascular death at the end of the follow-up for the ratios of l,25(OH)₂D/PTH and [l,25(OH)₂D]²/PTH were 0.741 (95% CI: 0.668-0.805), and 0.749 (95% CI: 0.677-0.812) respectively, which was similar to BNP (AUC 0.744 [(95% CI: 0.671-0.808]), but clearly higher than Gal-3 (AUC 0.660 [(95% CI: 0.583-0.731]) and 25(OH)D (AUC 0.529 [(95% CI: 0.451-0.606]; p<0.01) (Figure 1).

Kaplan-Meier survival curves for patients stratified with l,25(OH)₂D/PTH ratio diverged significantly (Log-rank test: p <0.001; Figures 2).

Table 4

Sensitivity Specificity P PV NPV

%

3 months 86.7 48.5 13.0 97.6

6 months 84.8 50.0 19.5 95.8

1 year 80.3 51.3 26.5 92.3

2 years 83.7 57.2 43.5 89.9

End 81.4 66,7 65.5 82.1

Based on the cut points derived from the ROC curves, Table 4 details over time, the capability of to classify the risks of adverse events. The Sensitivity and the Negative predictive values in prediction gradually declined, consistent with evolution of disease in previously lower-risk patients.

DISCUSSION

The main objective of this study was to investigate the prognostic value of the l,25(OH)₂D/PTH ratio for long-term CV death in patients with chronic HF. These results clearly demonstrated the relationship between decreased levels of l,25(OH)₂D and long-term CV death in HF and for the first time the value of the ratio of l,25(OH)₂D to PTH for the prognostication of HF patients. Vitamin D deficiency as measured by circulating 25(OH)D has previously been shown to be associated with cardiovascular diseases. To date, vitamin D's biologically active metabolite l,25(OH)₂D has not been routinely assessed. This is due, in part, to the lack of assays that are accurate and precise at low l,25(OH)₂D concentrations. The development of a novel, fully- automated, extraction-free l,25(OH)₂D immunoassay (DiaSorin), with precision and sensitivity superior to LC-MS/MS, has allowed the exploration of new biological relationships that until now were beyond the reach of current methodologies.

These data clearly demonstrate the relationship between decreased levels of l,25(OH)₂D and long-term CV death in HF. Most importantly these data show how the relationship between the hormones l,25(OH)₂D and PTH results in a potent biomarker for the prognosis of HF patients as Kaplan Meier curves identified early change in survival according to l,25(OH)₂D or the ratio. The association of l,25(OH)₂D levels and the risk of adverse outcomes in cardiovascular diseases was previously observed, however the ratio of l,25(OH)₂D to PTH is a novel biomarker not previously examined. The ratio of l,25(OH)₂D to PTH was at least equally predictive, if not more predictive, than other established biomarkers of HF severity such as Gal-3, BNP, NT-proBNP, and FGF-23. The l,25(OH)₂D/PTH ratio is clinically superior because of its higher AUC. The difference in AUC with a cohort of this size is very promising. In terms of specificity, it allows for the integration of interrelated confounders, which in turn allows for treatment based on two modulable factors.

The ratio of l,25(OH)₂D to PTH is a potent biomarker that combines the contribution of l,25(OH)₂D, (which, being the active metabolite, provides a truer value of vitamin D actions and sufficiency than circulating 25(OH)D in 25(OH)D deficient patients) and circulating PTH, which has been previously shown to be associated with mortality. In addition, studies have shown that PTH contributes to the pathophysiology and worsening of HF. Hyperparathyroidism was previously observed in patients with untreated and treated HF with reduced left ventricular ejection fraction with second and third generation immunoassays. In addition, PTH was shown to have several negative direct and indirect effects on the heart and cardiac cells (Tomaschitz A, et al. Cardiovasc Res 2012; 94(1): 10-19). Furthermore, increased circulating concentrations of PTH might stimulate adrenal aldosterone synthesis, initiating a vicious cycle between hyperparathyroidism and hyperaldosteronism and leading to more proinflammatory, pro-oxidant and pro-fibrotic actions.

FGF-23 is a key regulator of phosphorus homeostasis produced by osteocytes (Razzaque MS. Nat Rev Endocrinol 2009; 5(11):611-619). Previous studies have found significantly higher mortality in patients with FGF-23 levels >172 RU/ml). FGF-23 and FGF receptors are both expressed in the myocardium, and it was hypothesized that FGF-23 may have direct effect on the heart and participate to the physiopathology of cardiovascular diseases and HF. In cultured rat cardiomyocytes, FGF-23 was able to stimulate pathological hypertrophy through the activation of the calcineurin-NFAT pathway. Animal studies showed that mice given intra- myocardial or intravenous FGF-23 had an outcome of left ventricular hypertrophy. Interestingly, significant relationship between FGF-23 and PTH has been previously documented in chronic kidney disease and HF patients. As l,25(OH)₂D participates in the regulation of bone and mineral metabolism with PTH and FGF-23, the potential for significant diagnostic interrelation between l,25(OH)₂D, PTH and FGF-23 in the physiopathology of the cardio-renal syndrome related to HF is increasing. These data demonstrate that decreased l,25(OH)₂D levels are significantly related to HF severity and to the rise of serum PTH and FGF-23 levels. These findings also evidenced strong positive correlations between l,25(OH)₂D, BNP, NT-proBNP and Galectin-3. Decreased l,25(OH)₂D levels in HF patients were previously reported; however, the concentrations were obtained with less sensitive and less reliable assays.

The l,25(OH)₂D test used to make the determinations presented herein, in contrast to the tests for existing biomarkers, offers several advantages such as a broader availability on automated analytical platforms, significantly smaller sample volume, an ongoing effort of standardization, and the potential for accessibility and a short turn-around-time (65 minutes to first result). In addition, a pre-analytical extraction step normally required for other l,25(OH)₂D assays including LC-MS/MS, is not required by the instant assay. This simplification greatly diminishes the imprecision of the current assay compared to those previously used, especially at low concentrations. The effect on imprecision that results by combining values from two assays into a single ratio was also examined by using two different lots each of l,25(OH)₂D and PTH kits, and determining the ratio for the four resulting kit combinations. The coefficient of variation of the ratio was on average 2.8% for ratios >0.5 with a maximum CV of 6.3%.

The therapeutic rational for testing l,25(OH)₂D and its ratio to PTH in HF patients contributes to risk stratification, and also to treatment selection and monitoring of the efficiency of medical devices. Indeed, more tailored treatment selection might be guided by aiming for higher l,25(OH)₂D/PTH ratios, by aiming to increase l,25(OH)₂D in HF patients with lower l,25(OH)₂D levels with calcitriol supplementation (or other active metabolite) and/or to decrease PTH with other pharmacological treatments such as aldosterone blockers for example, known to prevent hyperparathyroidism and its consequences. Previous studies showed that l,25(OH)₂D supplementation has protective effects on myocardial fibrosis of diabetic rats, that it is effective in preserving endothelial function in hypertension, and it improved cardiac function in patients on hemodialysis that had controllable hyperparathyroidism. For example, one potential invention with significantly smaller risk to the subject would be to establish vitamin D sufficiency and then titrate aldosterone blockers (thereby suppressing PTH) according to beneficial ratio thresholds.

In conclusion, based on the data presented here, 1, 25 -dihydroxy vitamin D and its ratio to PTH are strong independent markers for cardiovascular death in chronic HF, comparable or better than currently used biomarkers such as BNP, Galectin-3, and FGF-23.

While this invention has been described in conjunction with the various exemplary embodiments outlined above, various alternatives, modifications, variations, improvements and/or substantial equivalents, whether known or that are or may be presently unforeseen, may become apparent to those having at least ordinary skill in the art. Accordingly, the exemplary embodiments according to this invention, as set forth above, are intended to be illustrative not limiting. Various changes may be made without departing from the spirit and scope of the invention. Therefore, the invention is intended to embrace all known or later-developed alternatives, modifications, variations, improvements and/or substantial equivalents of these exemplary embodiments.

List of References

Meijers WC, de Boer RA, van Veldhuisen DJ, et al. Biomarkers and low risk in heart failure.

Data from COACH and TRIUMPH. Eur J Heart Fail. 2015; 17(12): 1271-82.

Gruson D, Buglioni A, Burnett JC, Jr. PTH(l-84): Potential role in management of heart failure. Clin Chim Acta 2014; 433:290-6. doi: 10.1016/j.cca.2014.03.029. Epub;%2014 Apr l.:290-296.

Scialla JJ, Wolf M. Roles of phosphate and fibroblast growth factor 23 in cardiovascular disease. Nat Rev Nephrol 2014; 10(5):268-278.

Gruson D, et al. 1, 25 -Dihydroxy vitamin D to PTH(l-84) Ratios Strongly Predict Cardiovascular Death in Heart Failure. PlosOne 2015; 10(8):e0135427

Tomaschitz A, Ritz E, Pieske B et al. Aldosterone and parathyroid hormone: a precarious couple for cardiovascular disease. Cardiovasc Res 2012; 94(1): 10-19.

Razzaque MS. The FGF23-Klotho axis: endocrine regulation of phosphate homeostasis. Nat Rev Endocrinol 2009; 5(11):611-619.

Claims

1. A method for predicting or stratifying the risk of morbidity and/or mortality in a chronic heart failure patient, the method comprising:

(a) detecting and quantifying 1,25-dihydroxyvitamin D in a sample from the patient;

(b) detecting and quantifying parathyroid hormone in a sample from the patient; and

(c) calculating the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio;

wherein:

when the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is above a predetermined threshold, the patient is predicted or stratified not to have an increased risk of chronic heart failure morbidity and/or mortality; and

when the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is below a predetermined threshold, the patient is predicted or stratified to have an increased risk of chronic heart failure morbidity and/or mortality.

2. The method according to claim 1, wherein when the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is below the predetermined threshold, the chronic heart failure patient is stratified as having a high risk of chronic heart failure morbidity and/or mortality.

3. The method according to claim 1, wherein when the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is above the predetermined threshold, the chronic heart failure patient is stratified as having a low risk of chronic heart failure morbidity and/or mortality.

4. The method according to any of claims 1 to 3, wherein the predetermined threshold is comprised within the range of from 0.9 to 1.4.

5. The method according to claim 4, wherein the predetermined threshold is 1.06.

6. The method according to any of claims 1 to 5, wherein 1,25-dihydroxyvitamin D and/or parathyroid hormone are detected and quantified from blood, serum, plasma, saliva, lymph, urine or feces sample.

7. The method according to any of claims 1 to 6, wherein 1, 25 -dihydroxy vitamin D and/or parathyroid hormone are detected and quantified using an immunoassay.

8. The method according to claim 7, wherein the immunoassay is a chemiluminescent assay.

9. A method for predicting or stratifying the risk of morbidity and/or mortality in a chronic heart failure patient, the method comprising:

(c) calculating the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio;

wherein:

when the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is below a predetermined threshold, the patient is predicted or stratified to have an increased risk of chronic heart failure morbidity and/or mortality,

wherein 1,25-dihydroxyvitamin D is detected and quantified using an immunoassay which comprises:

(i) contacting the 1,25-dihydroxyvitamin D in the sample from the patient with a receptor protein comprising the Ligand Binding Domain of Vitamin D Receptor (VDR- LBD), thereby obtaining a first complex;

(ii) contacting said first complex with a capture moiety that specifically binds to a conformational epitope on said first complex, bud does not bind to either 1,25- dihydroxyvitamin D or VDR-LBD that is not bound in said first complex, thereby obtaining a second complex;

(iii) detecting and quantitating said second complex as an indication of the amount of 1,25 -dihydroxyvitamin D in the sample.

10. The method according to claim 9, wherein the capture moiety is a monoclonal antibody.

11. The method according to claim 9 or 10, wherein the capture moiety is immobilized on a solid support.

12. The method according to any of claims 9 to 11, wherein the immunoassay is a sandwich immunoassay.

13. The method according to claim 12, wherein the step (iii) of detecting and quantitating said second complex is carried out by means of a labeled anti-VDR-LBD detector antibody.

14. The method according to any of claims 9 to 13, wherein when the [1,25- dihydroxyvitamin D]/[parathyroid hormone] ratio is below the predetermined threshold, the chronic heart failure patient is stratified as having a high risk of chronic heart failure morbidity and/or mortality.

15. The method according to any of claims 9 to 13, wherein when the calculated ratio is above the predetermined threshold, the chronic heart failure patient is stratified as having a low risk of chronic heart failure morbidity and/or mortality.

16. The method according to any of claims 9 to 15, wherein the predetermined threshold is comprised within the range of from 0.9 to 1.4.

17. The method according to claim 16, wherein the predetermined threshold is 1.06.

18. The method according to any of claims 9 to 17, wherein 1,25-dihydroxyvitamin D and/or parathyroid hormone are detected and quantified from blood, serum, plasma, saliva, lymph, urine or feces sample.

19. The method according to any of claims 9 to 18, wherein parathyroid hormone is detected and quantified using an immunoassay.

20. The method according to claim 19, wherein the 1,25-dihydroxyvitamin D immunoassay and/or the parathyroid hormone immunoassay is a chemiluminescent assay.