AU2016207734A1 - Use of 1,25-dihydroxyvitamin D values in ratio with PTH as a prognostic biomarker - Google Patents

Abstract

Use of 1,25-dihydroxyvitamin D values in ratio with PTH as a prognostic biomarker The present invention relates to a method for predicting or stratifying the risk of worsening renal function (WRF) in a patient at risk of renal injury or affected by renal injury. Levels of 1,25-dihydroxyvitamin D (1,25(OH)

Description

WO 2016/113720 PCT/IB2016/050230 1

Use of 1,25-dihydroxyvitamin 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 worsening renal function (WRF) in a patient at risk of renal injury or in a patient affected by renal injury. Levels of 1,25-dihydroxy vitamin 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 the risk of worsening renal function.

BACKGROUND OF THE INVENTION

Progressive deterioration of renal function is common in patients with different diseases such as chronic heart failure (HF), chronic kidney disease (CKD) and metabolic syndrome, and is associated with unfavorable outcomes, which can be improved by timely interventions.

The cross talk between the diseased heart and kidney is a growing burden for health care systems as the incidence of HF and chronic kidney disease (CKD) has been steadily increasing and will further increase due to ageing of the general population and better treatment of acute cardiac and renal diseases. It has also been realized that the progressive development of worsening renal function (WRF) over time carries an increased risk of death and hospitalizations.

Identifying patients at risk of worsening renal function (WRF) is important for their clinical management, and might lead to less frequent hospitalizations and to the prevention of adverse outcomes. Early prediction and identification of patients at risk for future WRF may also be useful to optimize therapies and to improve outcomes. Circulating biomarkers may therefore provide a simple and objective means to predict deterioration in renal function in patients with chronic HF or other diseases such as chronic kidney disease (CKD) or IgA nephropathy. For instance, a common marker of renal injury is serum creatinine, which however is slowly affected by changes in renal function and is also WO 2016/113720 PCT/IB2016/050230 2 dependent on a plurality of different factors such as muscle mass, sex, race and age.

Disturbances of mineral metabolism, and in particular to the parathyroid hormone (PTH) / vitamin D axis, are characteristic of decreased renal function (1). Recent studies indicate that vitamin D-deficiency may promote or accelerate the progression of CKD. Cross-sectional studies have shown higher circulating levels of PTH and lower levels of vitamin D metabolites as CKD progresses (lower estimated glomerular filtration rate, eGFR). To the inventors’ knowledge, there are, however, scant reports on the ability of circulating markers of bone mineral metabolism to predict deterioration of renal function over time.

Therefore, the inventors examined the relation between two markers of the vitamin D/PTH axis and worsening of renal function (WRF) in a large cohort of patients with HF.

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 precluded evaluation of the prognostic value of this measurement.

Therefore, there is a need to identify biomarkers for predicting or stratifying the risk of worsening renal function (WRF) in a patient at risk of renal injury or in a patient affected by renal injury.

SUMMARY OF THE INVENTION

The present invention provides a method for predicting or stratifying the risk of worsening renal function (WRF) in a patient at risk of renal injury or in a patient affected by renal injury, using the level of 1,25(OH)2D in conjunction with the level of parathyroid hormone (PTH) to determine the ratio of l,25(OH)2D to PTH. The ratio value allows for risk prediction or stratification of worsening renal function in the 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. WO 2016/113720 PCT/IB2016/050230 3

Therefore, the invention provides a method for predicting or stratifying the risk of worsening renal function (WRF) in a patient at risk of renal injury or in a patient affected by renal injury, said method 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 worsening renal function, and when the ratio is below a predetermined threshold, the patient is predicted or stratified to have an increased risk of worsening renal function.

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

In various embodiments, l,25(OH)2D and/or PTH are determined from blood, plasma or serum 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 patient is predicted or stratified as having a high risk of worsening of renal function.

In another embodiment, when the l,25(OH)2D to PTH ratio is above the predetermined threshold, the patient is predicted or stratified as having a low risk of worsening renal function.

Preferably, the predetermined threshold in these embodiments is comprised within the range of from 0.92 to 1.8, more preferably the predetermined threshold is 0.92, 0.98 or 1.68.

In yet another embodiment, l,25(OH)2D is determined using an immunoassay which WO 2016/113720 PCT/IB2016/050230 4 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 1,25(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 skilled in the art will appreciate that any method of collecting reliable (sufficiently sensitive and accurate) 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 e.g. 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. WO 2016/113720 PCT/IB2016/050230 5

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 operator curve (ROC) and area under the curve (AUC) for the 1,25(OH)2D/PTH ratio with WRF

Figure 2: Kaplan-Meier curve for the first occurrence of WRF stratified by the [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; LVEF, left ventricular ejection fraction; GFR, glomerular fdtration rate; eGFR, estimated glomerular filtration rate; CV, cardiovascular; l,25(OH)2D, 1,25-dihydroxy vitamin D; WO 2016/113720 PCT/IB2016/050230 6 PTH, parathyroid hormone; ROC, receiver operating characteristics; AUC, area under the curve; CART, Classification and regression trees.

Impaired levels of 25-Hydroxy vitamin D (25(OH)D) (calcidiol) have previously been shown to be associated with an increased risk of various diseases including cardiovascular disease, hypertension, myocardial infarction, diabetes, cancer, reduced neuromuscular function, infectious and autoimmune disease. However, under conditions of vitamin D (cholcalciferaol) deficiency, this biomarker may not be as predictive as its biologically active metabolite 1,25-dihydroxy vitamin D (l,25(OH)2D) or calcitriol. l,25(OH)2D and PTH control calcium and phosphate homeostasis. The potential value of l,25(OH)2D testing as a significant predictor of renal injury or of worsening renal function in a cohort of patients, more particularly HF patients, was pursued in a patient model 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 allow for risk prediction or stratification of worsening renal function (WRF) in patients at risk of renal injury or in patients affected by renal injury.

Prior determinations for Vitamin D sufficiency have relied upon determining levels of circulating 25-Hydroxy vitamin 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 WO 2016/113720 PCT/IB2016/050230 7 spectrum of action, and has been associated with increased risks for various chronic infectious and autoimmune conditions, 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 cardiac, intestinal, immune, bone, neuronal degenerative, cancerous and diabetic conditions. While serum l,25(OH)2D values are not generally taken, it is considered that normal circulating levels of l,25(OH)2D in the United States are in the range of 19.9-79.3 pg/ml with a median of about 47.8 pg/ml (2).

Since the binding of l,25(OH)2D to the VDR-LBD is known to induce a conformational change, 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 unbound 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 specific to the VDR-LBD -l,25(OH)2D complex conformation.

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. WO 2016/113720 PCT/IB2016/050230 8 PTH is secreted by the parathyroid glands and acts to increase the concentration of calcium in the blood by binding to the parathyroid receptor (expressed at high levels in the bone and kidney) or to parathyroid hormone 2 receptor (expressed in the CNS, pancreas, testis and placenta). Further, PTH increases the activity of renal Ι-α-hydroxylase, which converts 25-hydroxy vitamin D to l,25(OH)2D to support endocrine function. Ι-α-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 5.72 to 45.4 pg/mL and 5.68 to 47.8 pg/mL in EDTA plasma and serum, respectively (3). 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).

The ratio of l,25(OH)2D to PTH is identified herein as being a biomarker for predicting or stratifying the risk of worsening renal function (WRF) in patients at risk of renal injury or in patients affected by renal injury.

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.

EXPERIMENTAL SECTION 1,25(ΟΗ)?Ρ/ΡΤΗ Biomarker of Renal Injury Materials and Methods

The cohort studied consisted of 1083 patients enrolled in a biomarker substudy of a randomized, double-blind, placebo controlled, multicenter study that enrolled 6975 patients with clinical evidence of chronic and stable HF (NYHA II-IV), irrespective of the cause and the level of left ventricular ejection fraction (LVEF). In relation to renal function, 180 patients of the cohort were normal (i.e., eGFR (mL/min/1.73 m2) >90) and the remainder had eGFR below 90. Venous blood samples were drawn on EDTA at randomization and after three months of follow-up. Patients rested supine for at least 15 min before blood sampling. Blood was centrifuged at 4°C within 10 minutes of draw and plasma aliquots WO 2016/113720 PCT/IB2016/050230 9 were shipped on dry ice to a central laboratory. Samples were stored at -70°C until assayed. In the analysis, worsening of renal function (WRF) was employed as the endpoint.

The plasma concentrations of 1,25-dihydroxy vitamin D (l,25(OH)2D) and PTH were assayed in a central laboratory in a blinded fashion and in a single batch. l,25(OH)2D was determined with a new fully automated and sensitive immunoassay that uses a recombinant fusion construct of the vitamin D receptor ligand binding domain for specific capture of l,25(OH)2D (DiaSorin, Saluggia, Italy). The limit of quantitation for this l,25(OH)2D assay is 5 pg/mL and the reference interval determined in healthy volunteers ranged between 19.9 and 79.3 pg/mL with a median of 47.8 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 5.5 to 48 (3) with a median of 15.3 pg/ml.

Serum creatinine was measured in local laboratories as part of a national quality control surveillance, at randomization and during follow-up after 1, 3, 6, 12, 24, 36, 48 and 60 months. Glomerular filtration rate (eGFR, mL/min/1.73m2) was estimated using the simplified modification of diet in renal disease (MDRD) formula. WRF was defined as the first increase in serum creatinine concentration >0.3 mg/dL and >25% at two consecutive measurements at any time during the study (4).

Statistical methods: Continuous variables were expressed as mean+SD if normally distributed or median [Ql- Q3], as appropriate; categorical variables were reported as absolute numbers and percentages.

Linear multilevel analysis was used to assess the association of baseline patient characteristics with decreasing baseline levels of the l,25(OH)2D to PTH ratio, which was transformed on a natural logarithmic scale. The model allowed consideration of variable patient characteristics as fixed effects whereas multiple clinical centers introduced random effects. A Cox proportional hazards model aiming to assess the independent prognostic value of WO 2016/113720 PCT/IB2016/050230 10 the l,25(OH)2D to PTH ratio on the occurrence of WRF was built, adjusting for the covariates that were statistically significant in the univariate analysis ( P<0.05). Similarly, multivariable Cox models were adopted for the secondary outcomes of the present analysis.

For all of the categorical variables, the proportionality of risk required by the Cox model was assessed using Schoenfeld residuals. The ratio was initially fitted as a single continuous measurement. Because there was clear evidence of non-linearity of risk detected by the restricted cubic splines technique (RCS), it was transformed with natural logarithms, hence satisfying the linearity assumption imposed by the Cox model.

To establish the incremental prognostic value of the l,25(OH)2D to PTH ratio on the occurrence of WRF, in addition to the conventional risk factors that emerged as statistically significant in the multivariable model, the category-free Net Reclassification Index (cfNRI) was calculated (5). A 2-sided P value of <0.05 was considered statistically significant. Statistical analyses were performed with SAS software, version 9.3 (SAS Institute, Inc., Cary, NC) and with the R program and the rms package (http://CRAN.R-project.org/package=rms).

Results

Study Population - Baseline characteristics: The distribution of baseline characteristics and laboratory values across the entire cohort according to 25(OH)D and l,25(OH)2D levels are displayed in Table 1.

Univariate and multivariable Cox proportional hazard models COX proportional hazard (CPH) analyses were carried out by entering biomarker concentration values into the models as loge-transformed variables. Table 2 shows the results of the Cox proportional hazard analysis for the association of WRF with baseline l,25(OH)2D alone, PTH alone and their ratio. WRF occurred at 189 [82-735] days after randomization (equivalent to 6.2 [2.7-24.1] months) (median [Q1-Q3]).

Table 1

Variable All N = 1130 No WRF N = 795 (70.4%) WRF N = 335 (29.6%) P Age (years) 66.8+10.8 66.2+11.1 68.1+9.8 0.004 Sex (% males) 917 (81.2) 638 (80.3) 279 (83.3) 0.23 BMIfkg/m*) 26.8+4.3 26.7+4.4 26.9+4.2 0.49 NYHA lll-IV (%) 290 (25.7) 191 (24.0) 99 (29.6) 0.052 Ischemic HF (%) 579 (51.2) 400 (50.3) 179 (53.4) 0.34 HR (bpm) 71.5+13.6 70.9+13.0 72.5+14.7 0.07 SBP (mmHci) 124.9+18.8 125.0+18.8 125+19.0 0.64 DBP (mmHg) 76.4+10.4 76.7+10.4 75.7+10.5 0.14 LVEF(%) 33.1+9.4 33.4+9.3 32.5+9.5 0.18 CVP > 6 cm H?0 (%) 89 (7.9) 61 (7.7) 28 (8.4) 0.70 Medical History Previous Ml (%) 495 (43.8) 338 (42.5) 157 (46.9) 0.18 Previous stroke (%) 53 (4.7) 36 (4.5) 17(5.1) 0.69 History of hypertension (%) 619 (54.8) 427 (53.7) 192 (57.3) 0.27 History of diabetes (%) 295 (26.1) 201 (25.3) 94 (28.1) 0.33 History of atrial fibrillation (%) 208 (18.4) 137 (17.2) 71 (21.2) 0.12 History of COPD (%) 210 (18.6) 138(17.4) 72 (21.5) 0.10 Laboratory parameters Serum creatinine (mg/dL) 1.20+0.42 1.16+0.37 1.29+0.45 <0.0001 eGFR (mL7min/1.73 m2) 68.6+23.5 71.0+23 63.7+22.9 <0.0001 Serum bilirubin (mg/dL) 0.84+0.55 0.86+0.61 0.80+0.36 0.07 Serum fibrinogen (mg/dL) 375+108 372+104 379+118 0.36 Serum cholesterol (mg/dL) 190+41 193+42 184+40 0.002 Serum LDL-cholesterol (mg/dL) 115+36 118+36 111+35 0.006 Serum HDL-cholesterol (mg/dL) 47+15 48+15 44+15 0.0004 Serum triglycerides (mg/dL) 123 [91-1741 123 [90-1761 124 [93-1681 0.90 Medical Therapy ACEi (%) 926 (82.0) 645 (81.1) 281 (83.9) 0.27 ARB (%) 196 (17.4) 134(16.9) 62(18.5) 0.50 Diuretics (%) 1045 (92.5) 719(90.4) 326 (97.3) <0.0001 Beta-blockers (%) 771 (68.2) 549 (69.1) 222 (66.3) 0.36 Spironolactone (%) 479 (42.4) 313(39.4) 166 (49.6) 0.002 Digitalis (%) 387 (34.3) 262 (33.0) 125 (37.3) 0.16 ASA (%) 569 (50.4) 421 (53.0) 148 (44.2) 0.007 Nitrates (%) 358 (31.7) 249 (31.3) 109 (32.5) 0.69 WO 2016/113720 PCT/IB2016/050230

Amiodarone (%) 230 (20.4) 141 (17.7) 89 (26.6) 0.0008 Randomization to n-3 PUFA (%) 569 (50.4) 407 (51.2) 162 (48.4) 0.38 Randomization to rosuvastatin (%) 334 (50.7) 235 (50.5) 99 (51.0) 0.91 Biomarkers 1,25(OH)? vitamin D (pg/mL) 31.3 (23.0-42.21 32.2 (24.1-43.11 28.7 [21.4-38.61 0.0001 PTH (1-84) (pg/mL) 33.7 [24.3-49.31 32.5 [23.6-46.41 36.3 [26.2-53.41 0.0008 1,25(OH)? vitamin D/PTH 0.89 [0.55-1.451 0.98 [0.60-1.531 0.73 [0.46-1.261 <0.0001 Blood samples drawn in winter (No., %) 328 (29.0) 235 (29.6) 93 (27.8) 0.26 WO 2016/113720 PCT/IB2016/050230 WO 2016/113720 PCT/IB2016/050230 13

Table 2A

Univariate No. HR [95%CI] P 1,25(OH)2D 1098 0.61 [0.49-0.75] <0.0001 PTH (1-84) 1099 1.57 [1.28-1.93] <0.0001 1,25(OH)2D/PTH 1067 0.60 [0.52-0.70] <0.0001

When multivariable COX analyses were performed, the following variables were entered into the Cox multivariate models: l,25(OH)2D/PTH (loge-transformed), age, eGFR (MDRD equation), NYHA class, serum concentrations of total cholesterol and bilirubin, prescriptions of diuretics, spironolactone, aspirin or amiodarone.

Table 2B

Multivariate No. HR [95%CIJ P 1,25(OH)2D 1043 0.76 [0.59-0.97] 0.03 PTH (1-84) 1040 1.20 [0.95-1.50] 0.12 1,25(OH)2D/PTH 1012 0.75 [0.62-0.90] 0.003

Variables significantly associated with WRF in the multivariable models:

With l,25(OH)2D: serum cholesterol (p=0.0003), amiodarone (p=0.0005), aspirin (p=0.01), diuretics (p=0.02), serum bilirubin (p=0.02), spironolactone (p=0.04).

With PTH: amiodarone (p=0.0003), serum cholesterol (p=0.0004), aspirin (p=0.02), diuretics (p=0.03), spironolactone (p=0.03), serum bilirubin (p=0.04).

With l,25(OH)2D/PTH: serum cholesterol (p=0.0001), amiodarone (p=0.0008), aspirin (p=0.01), diuretics (p=0.02), spironolactone (p=0.03), serum bilirubin (p=0.04), eGFR (0.05).

Prognostic accuracy of baseline 1.25(OH)?D/PTH for WRF ROC curves a) ROC curves of baseline l,25(OH)2D/PTH with WRF.

In ROC analysis, the area under the curve (AUC) criterion was applied to WRF at the end of the 3.9 year follow-up (Figure 1). The receiver operator curve depicted in Figure 1 for WO 2016/113720 PCT/IB2016/050230 14 WRF at 3.9 years follow up to baseline randomization discloses an AUC of 60%, with specificity of 54% and sensitivity of 64% using the optimal l,25(OH)2D / 1-84 PTH ratio threshold of 0.92.

Table 3 AUROC ± SD P Specificity Sensitivity Optimal cut-off 1,25(OH)2D/PTH 0.60 ±0.02 <0.0001 0.54 0.64 0.92

Kaplan-Meier curves for the first occurrence of WRF according to the l,25(OH)2D to PTH ratio are presented in Figure 2. The Kaplan Meier curves in Figure 2 depict the ability of the l,25(OH)2D/l-84PTH ratio to discriminate amongst HF subjects those which have a significantly greater likelihood to both develop WRF and whose WRF will progress more quickly. With advancing time post randomization, those HF subjects whose baseline l,25(OH)2D/l-84PTH ratio were below the 0.92 threshold developed WRF at a faster rate than those whose ratio were > 0.92. b) The maximal negative predictive value (NPV= 0.82) is observed at a ratio of l,25(OH)2D to PTH of 1.68, with a sensitivity of 0.90, a specificity of 0.21 and a positive predictive value PPV= 0.32. A total of 187 patients (17.5%) have a ratio above the level of 1.68. At this cut-off, the contingency table for the occurrence of WRF is as follows: N W RF Yes (positive) No (negative) 1,25(OH)2 D/PTH <1.68 (positive) 285 595 >1.68 (negative) 33 154

True positive: 285/1067= 26.7%

False positive: 595/1067=55.8%

False negative: 33/1067=3.1%

True negative: 154/1067=14.4%

Test performance at the cutoff of the ratio 1.68 (maximal NPV): PPV = TP/(TP + FP) = A/(A+B) = 285 / (285 + 595) = 0.3239 NPV = TN/(TN + FN) = D/(D+C) = 154 / (154 + 33) = 0.8235 Sensitivity = TP/(TP + FN) = A/(A+C) = 285 / (285 + 33) = 0.8962 WO 2016/113720 PCT/IB2016/050230 15

Specificity = TN/(TN + FP) = D/(D+B) = 154 / (154 + 595) = 0.2056

Statistic Value ( % ) Sensitivity 89.6 Specificity 20.6 Negative Predictive Value (NPV) 82.4 Positive Predictive Value (PPV) 32.4 c) A CART approach was then used to define the best cut-off value of the l,25(OH)2D to PTH ratio to predict WRF.

Classification and regression trees (CART) is a model-free approach used to find the best splitting criterion. This method, a form of recursive partitioning, developed on 0.9 of the data allows to validate best on the remaining 0.1 of the data.

At the optimal cut-off of 0.98, the contingency table for the occurrence of WRF is as follows: N W RF Yes (positive) No (negative 1,25(OH)2 D/PTH <0.98 (positive) 217 372 >0.98 (negative) 101 377

True positive: 217/1067= 20.3%

False positive: 372/1067=34.9%

False negative: 101/1067=9.5%

True negative: 377/1067=35.3%

Test performance at the cutoff of the ratio > 0.98 (best cut-off identified with CART method) PPV = TP/(TP + FP) = A/(A+B) = 217 / (217 + 372) = 0.3684 NPV = TN/(TN + FN) = D/(D+C) = 377 / (377 + 101) = 0.7887

Sensitivity = TP/(TP + FN) = A/(A+C) = 217 / (217 + 101) = 0.6823 Specificity = TN/(TN + FP) = D/(D+B) = 377 / (377 + 372) = 0.5033 WO 2016/113720 PCT/IB2016/050230 16

Statistic Value (% ) Sensitivity 68.2 Specificity 50.3 Negative Predictive Value (NPV) 78.9 Positive Predictive Value (PPV) 36.8

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.

Discussion

The present inventors found that the ratio of circulating l,25(OH)2D and PTH, two hormones involved in bone-mineral metabolism, can surprisingly predict deterioration of renal function better than either marker alone. These results were obtained in a large, representative cohort of patients with HF, some of which were affected by renal injury, enrolled in a controlled, multicenter clinical trial.

Early prediction and identification of patients at risk for WRF may be useful to optimize therapies, and to improve outcomes. The search for new markers of changes in renal function is currently very active. They should be more sensitive and specific to early changes in renal function than serum creatinine, which is slowly affected and can be confounded by muscle mass and anthropometric factors.

In the present study, the inventors showed that a low circulating ratio of l,25(OH)2D to PTH predicted future episodes of WRF in patients. Although this ratio was strongly associated with concomitant serum creatinine levels, its prognostic value was independent of eGFR estimated from creatinine levels, suggesting a net contribution of vitamin D WO 2016/113720 PCT/IB2016/050230 17 metabolism to the prediction of WRF.

In conclusion, the ratio of l,25(OH)2D to PTH is a new, powerful indicator of future risk of deterioration of renal function in patients at risk of renal injury and affected by renal injury.

List of References 1. Evenepoel P, Rodriguez M, Ketteler M 2014 Laboratory Abnormalities in CKD-MBD: Markers,

Predictors, or Mediators of Disease? Semin Nephrol 34(2), 151-163.

2. LIAISON® XL 1,25 Dihydroxyvitamin D Assay (REF 310980, REF 310981) IFU (Instructions for Use) 3. LIAISON® 1-84 PTH Assay (REF 310630, REF 310631) IFU (Instructions for Use) 4. Damman K, Tang WH, Testani JM, McMurray JJ 2014 Terminology and Definition of Changes to Renal Function in Heart Failure Eur Heart J 35(48), 3413-3416. 5. Pencina MJ, DAgostino RB, Sr., Steyerberg EW 2011 Extensions of Net Reclassification Improvement Calculations to Measure Usefulness of New Biomarkers Stat Med 30, 11-21.

Claims (20)

1. A method for predicting or stratifying the risk of worsening renal function (WRF) in a patient at risk of renal injury or affected by renal injury, the method comprising: (a) detecting and quantifying 1,25-dihydroxy vitamin 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 worsening renal function, 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 worsening renal function.

2. The method according to claim 1, wherein when the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is below the predetermined threshold, the patient is predicted or stratified as having a high risk of worsening renal function.

3. The method according to claim 1, wherein when the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is above the predetermined threshold, the patient is predicted or stratified as having a low risk of worsening renal function.

4. The method according to any of claims 1 to 3, wherein the predetermined threshold is comprised within the range of from 0.92 to 1.8.

5. The method according to claim 4, wherein the predetermined threshold is 0.92, 0.98 or 1.68.

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 or plasma samples.

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 worsening renal function (WRF) in a patient at risk of renal injury or affected by renal injury, the method comprising: (a) detecting and quantifying 1,25-dihydroxy vitamin 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 worsening renal injury, 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 worsening renal function, wherein 1,25-dihydroxy vitamin D is detected and quantified using an immunoassay which comprises: (i) contacting the 1,25-dihydroxy vitamin 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, but 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-dihydroxy vitamin 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 patient is predicted or stratified as having a high risk of worsening renal function.

15. The method according to any of claims 9 to 13, wherein when the [1,25-dihydroxyvitamin D]/[parathyroid hormone] ratio is above the predetermined threshold, the patient is predicted or stratified as having a low risk of worsening renal function.

16. The method according to any of claims 9 to 15, wherein the predetermined threshold is comprised within the range of from 0.92 to 1.8.

17. The method according to claim 16, wherein the predetermined threshold is 0.92, 0.98 or 1.68.

18. The method according to any of claims 9 to 17, wherein 1,25-dihydroxy vitamin 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-dihydroxy vitamin D immunoassay and/or the parathyroid hormone immunoassay is a chemiluminescent assay.