Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
  • G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
  • G01N33/541—Double or second antibody, i.e. precipitating antibody
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/34—Genitourinary disorders
  • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

• 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.
• WRF worsening renal function
• Levels of 1,25-dihydroxy vitamin D (l,25(OH) 2 D) 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.
• HF chronic heart failure
• CKD chronic kidney disease
• metabolic syndrome 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.
• 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.
• CKD chronic kidney disease
• IgA IgA nephropathy
• 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.
• 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 l,25(OH) 2 D in conjunction with the level of parathyroid hormone (PTH) to determine the ratio of l,25(OH) 2 D to PTH.
• the ratio value allows for risk prediction or stratification of worsening renal function in the patient.
• PTH parathyroid hormone 1-84
• PTH 1-84 parathyroid hormone 1-84
• WRF renal function
• 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) 2 D in a sample from the patient; (b) detecting and quantifying PTH in a sample from the patient; and (c) calculating the l,25(OH) 2 D 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) 2 D 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.
• l,25(OH) 2 D and/or PTH are determined from blood, plasma or serum samples from the patient. In these and other embodiments, l,25(OH) 2 D and/or PTH are determined using an immunoassay. In particular embodiments, the immunoassay is a chemiluminescent assay.
• the patient when the ratio of l,25(OH) 2 D to PTH is below the predetermined threshold, the patient is predicted or stratified as having a high risk of worsening of renal function.
• the patient when the l,25(OH) 2 D to PTH ratio is above the predetermined threshold, the patient is predicted or stratified as having a low risk of worsening renal function.
• 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.
• l,25(OH) 2 D is determined using an immunoassay which comprises (i) contacting the l,25(OH) 2 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 l,25(OH) 2 D 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) 2 D in the sample.
• VDR-LBD Ligand Binding Domain of Vitamin D Receptor
• the capture moiety is a monoclonal antibody.
• the capture moiety is immobilized on a solid support.
• the immunoassay of the claimed method is a sandwich immunoassay.
• step (iii) of detecting and quantitating said second complex is carried out by means of a labeled anti- VDR-LBD detector antibody.
• FIG. 1 Receiver operator curve (ROC) and area under the curve (AUC) for the l,25(OH) 2 D/PTH ratio with WRF
• HF Heart Failure
• NYHA New York Heart Association
• PPV Positive Predictive Value
• NPV Negative Predictive Value
• LVEF left ventricular ejection fraction
• GFR glomerular filtration rate
• eGFR estimated glomerular filtration rate
• CV cardiovascular
• l 25(OH) 2 D, 1,25-dihydroxy vitamin D
• PTH parathyroid hormone
• ROC receiver operating characteristics
• AUC area under the curve
• CART Classification and regression trees.
• the present study was designed using a new, fully-automated l,25(OH) 2 D assay with improved analytical performance, sensitivity, and reliability.
• the inventors tested the hypothesis that levels of l,25(OH) 2 D 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.
• WRF worsening renal function
• 25(OH)D 25-Hydroxyvitamin D
• cholecalciferol vitamin D
• 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.
• circulating 25(OH)D is transported to the kidneys where it is converted to its active form l,25(OH) 2 D (calcitriol).
• l,25(OH) 2 D 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) 2 D circulates in the blood bound to the vitamin D binding protein, and enters target cells where the l,25(OH) 2 D is made available to bind to the vitamin D receptor (VDR).
• VDR vitamin D receptor
• This ligand/receptor complex readily translocates across the nuclear membrane to act as a transcription factor.
• l,25(OH) 2 D is now known to have a broader 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.
• l,25(OH) 2 D 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) 2 D values are not generally taken, it is considered that normal circulating levels of l,25(OH) 2 D 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).
• immunoassay methods for detecting total l,25(OH) 2 D 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) 2 D, in order to selectively discriminate the VDR-LBD/l,25(OH) 2 D complex from either unbound l,25(OH) 2 D or unbound VDR-LBD, as described in WO2014114780.
• the capture moiety of the method of the invention is a monoclonal antibody specific to the VDR-LBD - l,25(OH) 2 D complex conformation.
• the detection of the captured VDR- LBD/l,25(OH) 2 D 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) 2 D complex captured by the capture moiety.
• the detector molecule is an antibody directed to an epitope on the VDR-LBD/l,25(OH) 2 D complex which is different from the epitope recognized by the capture moiety.
• the l,25(OH) 2 D immunoassay of the method of the invention is a sandwich immunoassay, more preferably a chemiluminescence immunoassay.
• the capture antibody may be immobilized on a solid support.
• 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 (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 1-a-hydroxylase, which converts 25 -hydroxy vitamin D to l,25(OH) 2 D to support endocrine function. 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 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) 2 D to PTH in normal individuals is approximately 2.7 (range of 1.2-9.1).
• the ratio of l,25(OH) 2 D 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.
• 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).
• LVEF left ventricular ejection fraction
• 180 patients of the cohort were normal (i.e., eGFR (mL/min/1.73 m ) >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.
• l,25(OH) 2 D The plasma concentrations of 1,25-dihydroxy vitamin D (l,25(OH) 2 D) and PTH were assayed in a central laboratory in a blinded fashion and in a single batch.
• l,25(OH) 2 D 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) 2 D (DiaSorin, Saluggia, Italy).
• the limit of quantitation for this l,25(OH) 2 D 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/1.73m ) 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).
• a Cox proportional hazards model aiming to assess the independent prognostic value of the l,25(OH) 2 D 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.
• 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) 2 D levels are displayed in Table 1.
• 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]).
• 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 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) 2 D / 1-84 PTH ratio threshold of 0.92.
• Kaplan-Meier curves for the first occurrence of WRF according to the l,25(OH) 2 D 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.
• 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.
• 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.
• the present inventors found that the ratio of circulating l,25(OH) 2 D and PTH, two hormones involved in bone-mineral metabolism, can surprisingly predict deterioration of renal function better than either marker alone.
• the ratio of l,25(OH) 2 D 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.
• LIAISON ® XL 1,25 Dihydroxyvitamin D Assay (REF 310980, REF 310981) IFU (Instructions for Use)
• LIAISON ® 1-84 PTH Assay (REF 310630, REF 310631) IFU (Instructions for Use) 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.
• Pencina MJ D'Agostino RB, Sr., Steyerberg EW 2011 Extensions of Net Reclassification Improvement Calculations to Measure Usefulness of New Biomarkers Stat Med 30, 11-21.

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• 2016
  • 2016-01-18 EP EP16707951.6A patent/EP3245515A1/de not_active Withdrawn
  • 2016-01-18 WO PCT/IB2016/050229 patent/WO2016113719A1/en not_active Ceased
  • 2016-01-18 WO PCT/IB2016/050230 patent/WO2016113720A1/en not_active Ceased
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