HK1124391A - The use of bnp-type peptides for predicting the need for dialysis - Google Patents

Description

use of BNP-type peptides for predicting the need for dialysis

The present invention relates to diagnosing the risk of developing a need for dialysis, in particular in patients suffering from renal disorders. More particularly, the present invention relates to predicting the need for dialysis and/or the time until dialysis is needed. The present invention thus also provides risk stratification which patients will need to be closely monitored and/or benefit from initiating early intensive therapy.

One goal of modern medicine is to provide personalized or individualized treatment regimens. These treatment regimens take into account the individual needs or risks of the patient, for example, by selecting a particular treatment or monitoring regimen.

Dialysis is a type of renal replacement therapy used to provide an artificial replacement for the loss of renal function due to renal failure. It is primarily a life support therapy and is generally not curative for any kidney disease. Dialysis can be used in very severe patients who have suddenly lost their kidney function (acute renal failure) or in fairly stable patients whose kidney function is continuously worsening (chronic renal failure, chronic kidney disease) until dialysis has to be started to treat uremia (end stage renal failure, end stage chronic kidney disease).

In this context, it should be noted that survival is likely in the presence of chronically impaired renal function or even with only one kidney. Chronic renal failure will occur only when the amount of functional renal tissue is substantially reduced. Such decompensation of renal function can affect the function of many other organs and naturally leads to death from uremia. In patients with acute or chronic decompensation with severe uremia, immediate renal replacement therapy, such as dialysis or renal transplantation, is required. However, in contrast to the chronic deterioration and progressive decompensation of renal function that is commonly observed, renal failure can occur very suddenly and unexpectedly due to infection, sepsis, or toxic side effects of various medical therapies, even in patients that were previously thought to still have stable renal function. Despite established laboratory values for assessing renal insufficiency (see below), it would be highly desirable to have readily diagnostically useful tools and methods, such as biomarkers, to identify patients at risk of developing dialysis.

Most physicians use plasma concentrations of creatinine, urea, cystatin C, and electrolytes to determine renal function. These measurements are sufficient to determine whether a patient has renal disease. Unfortunately, blood urea nitrogen and creatinine will not fall outside the normal range until 60% of total renal function is lost.

In renal patients, an estimate of Glomerular Filtration Rate (GFR) is used to assess renal function. GFR was calculated by comparing the urine creatinine level to the blood test results. It yields a more accurate indication of renal status. GFR is expressed in mL/min (milliliters per minute). For most patients, a GFR of greater than 60mL/min is sufficient. However, if GFR drops significantly from previous test results, this may be an early indicator of renal disease requiring medical intervention.

Cystatin C measurements correlate more closely with creatinine than GFR. The serum cystatin C reference value was significantly higher in men than in women. Furthermore, serum cystatin C increases with age above 60 years. In older individuals, there were no gender-related differences in the reference values. Cystatin C is thought to be independent of body composition, however, lean body mass affects the level of cystatin C and when this variable is considered, the prediction of the GFR based on cystatin C is improved.

However, although these markers are currently used to determine the current state of kidney function, they are simply not sufficient to diagnose the risk of developing a need for dialysis.

Sanchez et al (2004) have studied pre-and intra-operative predictors of the need for renal replacement therapy in patients who have received liver transplants. They described that pre-operative serum creatinine greater than 1.9mg/dL, pre-operative blood urea nitrogen greater than 27mg/dL, intensive care unit stays greater than 3 days, and end-stage liver disease model scores greater than 21 were significant (Sanchez EQ, Gonwa TA, Levy MF, Goldstein RM, et al (2004) advanced and periodic predictions of the recent for new reproduction thermal after surgery vertical translation. transfer, vol.78(7), pp.1048-54). However, this combination of indicators applies only to liver transplant recipients and cannot be easily generalized.

Several putative markers such as MCP-1, ADMA, biopsy (e.g., morphometric determination of chronic kidney injury in lupus nephritis) have been investigated for the prediction of dialysis need.

BNP-type peptides, such as Brain Natriuretic Peptide (BNP) and/or its N-terminal propeptide fragment (NT-proBNP), and their use as molecular or biochemical markers for diagnosing certain disorders are likewise known. In WO 02/089657, it has been proposed to measure Brain Natriuretic Peptide (BNP) to diagnose myocardial infarction. In WO 02/083913, it has been proposed to use BNP to predict recent morbidity or mortality in patients with congestive heart failure, myocardial infarction, ST-elevated myocardial infarction or non-ST elevated acute coronary syndrome. It is suspected that plasma levels of NT-proBNP are also affected by the presence of chronic kidney disease, suggesting that NT-proBNP may not be a satisfactory marker of renal function.

Thus, currently, only a limited number of candidate tools are described in the prior art for assessing the potential need for dialysis.

Therefore, there is still a need to improve the risk of developing the need for dialysis for diagnosis and to overcome the drawbacks of the prior art. In particular, there is a need to provide reliable and efficient tools for diagnosing the risk of developing a need for dialysis.

The object of the present invention is achieved by a method for diagnosing and predicting the risk and time to develop a need for dialysis in a patient with chronic renal failure, said method comprising the steps of:

a) measuring the level of a BNP-type peptide or variant thereof in a sample from the patient,

b) diagnosing and predicting the risk of requiring dialysis by comparing the measured level of the BNP-type peptide or variant thereof with at least one reference level.

The invention also includes the step of obtaining a sample of a body fluid or tissue from a patient. Preferably, the level in a body fluid or tissue sample of the patient is measured.

The present invention provides methods and means, in particular markers, which allow diagnosing the risk of developing a need for dialysis. The invention therefore also allows predicting the need for dialysis and/or the time until the need for dialysis is developed. More specifically, it has been found in the context of the present invention that measured levels of BNP-type peptides can indicate the risk of developing a need for dialysis. The methods and tools provided herein are simple, quick, and inexpensive, and are suitable for use by practitioners and more specialized doctors, clinics, or laboratories. The invention also provides corresponding uses of any of the markers, tools and methods according to the invention.

In the course of the present invention, it has been found that the level of BP-type peptide in a patient may be indicative of the risk of developing a need for dialysis. Furthermore, it has been found that the level of BP-type peptides allows predicting whether the need for dialysis will occur rather early or rather late. These findings are quite unexpected, as initially it was thought that the levels of BP-type peptides would be strongly influenced by the presence or absence of heart disease, and thus it was thought that BP-type peptides could not predict the risk of developing a need for dialysis.

In particular, the invention also allows for an early diagnosis of the risk (and/or prognosis) of the development of a need for dialysis, among other things. Thus, the present invention will allow for the identification of patients at increased risk for renal function decompensation. Thus, it is possible to regulate and optimize the renal protection therapy earlier than before. The invention thus allows to retain more nephrons and possibly delay the need for dialysis by starting therapy earlier and/or in a more intensive way in patients already identified as having an elevated risk of dialysis need.

Thus, the invention also allows for appropriate concomitant treatment or monitoring depending on the risk schedule. The present invention therefore also provides for risk stratification, particularly with respect to which patients will need closer monitoring and/or stratification of patients who benefit the most from initiating early intensive therapy.

As already mentioned, survival is very likely in the case of impaired renal function or even only one kidney. For example, in humans, there is more renal tissue than is required for survival. However, if the amount of functional kidney tissue is greatly reduced, chronic renal failure may occur or renal dysfunction may lead to severe syndromes. In these cases, renal replacement therapy (e.g., dialysis or renal transplantation) is required.

The present invention is particularly beneficial to medical practitioners and physicians who are generally unable to obtain specialized equipment and are inexperienced at adequately diagnosing and evaluating renal dysfunction. However, the invention is also particularly useful for nephrologists and/or diabetes specialists. Other examples include a renal or diabetic clinic or department.

The invention will be particularly useful in the context of any renal disorder. Renal disorders are known to those skilled in the art. According to the present invention, the term "renal disorder" is considered to relate to any disease, injury or dysfunction of the kidney or to influence the kidney, more specifically to influence the ability of the kidney to waste removal and/or ultrafiltration.

Examples of renal disorders include congenital disorders or acquired disorders. The invention is particularly applicable to acquired renal disorders. Examples of congenital renal disorders include congenital hydronephrosis, congenital obstruction of the urinary tract, replicating ureters, horseshoe kidney, polycystic kidney disease, renal dysplasia, unilateral small kidney. Examples of acquired renal disorders include diabetic or indolent renal disease, glomerulonephritis, hydronephrosis (enlargement of one or both kidneys due to obstruction of urine flow), interstitial nephritis, kidney stones, kidney tumors (e.g., nephroblastoma and renal cell carcinoma), lupus nephritis, minimally altered renal disease, nephrotic syndrome (glomeruli have been damaged such that large amounts of protein in the blood enter the urine). Other common features of nephrotic syndrome include swelling, low serum albumin and high cholesterol), pyelonephritis, renal failure (e.g., acute renal failure and chronic renal failure).

More specifically, the present invention may be used for diagnosing the risk of developing a concurrent heart disease, such as hypertension, chronic heart failure, chronic ischemic heart disease or diabetes as described above, in a patient.

The renal disorder can be diagnosed by any known and deemed suitable means. In particular, renal function can be assessed by Glomerular Filtration Rate (GFR). GFR can be calculated, for example, by the Cockgroft-Gault or MDRD formula (Levey 1999, Annals of Internal Medicine, 461-. GFR is the volume of fluid filtered from the glomerular capillaries to the renal capsule per unit time. GFR was initially estimated by injection of inulin into plasma (GFR could never be determined, all calculations from the Cockgroft Gault formula such as the MDRD formula only yield an estimated rather than a "true" GFR). Because inulin is not reabsorbed by the kidneys after glomerular filtration, its rate of excretion is directly proportional to the rate at which water and solutes are filtered through the glomerular filter. However, in clinical practice, GFR is measured by creatinine clearance. Creatinine is an endogenous molecule, synthesized in the body, that is freely filtered by the glomerulus (and is secreted by the renal tubules in very small amounts). Creatinine clearance (CrCl) is therefore a fairly close approximation of GFR. GFR is typically recorded in milliliters per minute (mL/min). The normal range of GFR for males is 97 to 137mL/min, and the normal range of GFR for females is 88 to 128 mL/min.

If the GFR has decreased below a critical threshold, which allows the removal of toxic concentrations of blood uremia (typically at GFR, CrCl < 10-15mL/min, end stage renal failure), renal replacement therapy is also indicated, depending on other clinical circumstances such as the clinical condition of the patient, e.g., diagnosis of renal disorders by any known and deemed appropriate means. Specifically, renal function was assessed by GFR. One of the first implications of renal disorders is the presence of protein in the urine (microalbuminuria or macroalbuminuria), which can be assessed by a simple scale. The most common blood test used to date is still creatinine, which is recognized to be of low accuracy.

If GFR has decreased to a very low level (end stage renal failure), renal replacement therapy, e.g., dialysis or kidney transplantation, is required.

Preferably, a GFR of less than 10mL/min indicates dialysis is required and a GFR of less than 6mL/min indicates immediate dialysis is required. Dialysis is also preferably required if the patient has a GFR of less than 15mL/min and exhibits at least one of the following clinical conditions: uremic symptoms or signs, diuretic resistance fluid excess, insufficiently controlled blood pressure or evidence of malnutrition.

The term "dialysis" is known to the person skilled in the art. In particular, dialysis is a type of renal replacement therapy that can be used to provide an artificial replacement for the loss of renal function due to renal failure. It is primarily a life support therapy and is generally not curative for any kidney disease. Dialysis can be used in very ill patients who have suddenly lost their renal function (acute renal failure) or in fairly stable patients who have permanently lost their renal function (end-stage renal failure). When healthy, the kidneys remove waste products (e.g., potassium, acid, and urea) from the blood and remove excess liquid in the form of urine. Dialysis treatment can duplicate both functions. Thus, the term "dialysis" according to the present invention preferably relates to waste removal and/or ultrafiltration (liquid removal). More specifically, the term "dialysis" relates to waste removal, most specifically to a combination of waste removal and ultrafiltration.

The term "need for dialysis" is known to the person skilled in the art. In particular, according to the present invention, the term is considered to relate to the need for any type of renal replacement therapy, including, for example, dialysis, renal transplantation, and renal tissue transplantation. More specifically, the term "need for dialysis" relates to the need for either type of renal therapy, which has an effect on waste removal and ultrafiltration comparable to dialysis, using, for example, online HDF of Fresenius Medical Care (germany).

The present invention utilizes certain biochemical or molecular biological markers. The terms "biochemical marker" and "molecular marker" are known to those skilled in the art. In particular, biochemical or molecular markers are gene expression products that are differentially expressed (i.e., up-regulated or down-regulated) in the presence or absence of certain conditions, diseases, or complications. Generally, a molecular marker is defined as a nucleic acid (e.g., mRNA), while a biochemical marker is a protein, polypeptide, or peptide. The level of a suitable biochemical or molecular marker may be indicative of the presence or absence of the condition, disease or complication, thereby allowing diagnosis.

The invention makes use, inter alia, of BNP-type peptides as biochemical markers. Any combination of BNP-type peptides is also contemplated as biochemical markers in the context of the present invention. Advantageously, it has been found in the studies of the present invention that BNP-type peptides and in particular NT-probnp are accurate, effective and statistically independent predictors of the risk of developing a need for dialysis in terms of GFR.

BNP-type peptides comprise pre-pro BNP, NT-pro BNP and BNP.

The prepropeptide (134 amino acids in the case of pre-pro BNP) comprises a short signal peptide which is enzymatically cleaved to release the propeptide (108 amino acids for pro BNP). The pro peptide is further cleaved into an N-terminal pro peptide (NT-pro peptide, 76 amino acids for NP-proBNP) and an active hormone (32 amino acids in the case of BNP).

Preferred BNP-type peptides according to the invention are pro-BNP, NT-pro-BNP, BNP and variants thereof. BNP is an active hormone and has a shorter in vivo half-life than possible inactive NT-probnp.

Using NT-proBNP, the pre-analysis is more stable, allowing easy transport of the sample to the central laboratory (Mueller T, Gegenhuber A, Diplinger B, Poelz W, Haltmayer M.Long-term stability of endogenous B-type biological peptide (BNP) and amino terminal proBNP (NT-proBNP) in free zen plasma samples. clin ChemLab Med 2004; 42: 942-4.). The blood samples can be stored at room temperature for several days or can be mailed or transported without recovery losses. In contrast, 48 hours of BNP storage at room temperature or at 4 ℃ resulted in a concentration loss of at least 20% (Mueller T, Gegenhuber A, et al, Clin Chem Lab Med 2004; 42: 942-4, supra; Wu AH, Packer M, Smith A, Bijou R, Fink D, Mairj, Walllentin L, Johnston N, Feldcamp CS, havestick DM, Ahnadi CE, Grant A, Desperse N, Bluestein B, Ghani F. analytical and clinicaleucation of the Bayer ADVIA centre automated B-type natural peptide assays with a heart failure: a multisite study. Clin Chem 2004; 50: 867-73).

Either measurement of active or inactive forms may be advantageous depending on the time course of interest and available analytical equipment or storage conditions. The most preferred BNP-type peptides according to the invention are NT-probnp and variants thereof.

The term "variant" in this context relates to a peptide which is substantially similar to said peptide. The term "substantially similar" is known to those skilled in the art. In particular, the variant may be an isoform or allele that exhibits a short amino acid exchange compared to the amino acid sequence of the most prevalent peptide isoform in the human population. Preferably, such substantially similar peptides have a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% to the most prevalent isoform of the peptide. Substantially similar are also degradation products, e.g., proteolytic degradation products, which are still recognized by the diagnostic tool or by ligands directed against the respective full-length peptide. The term "variant" is also intended to refer to splice variants.

The term "variant" also relates to post-translationally modified peptides, such as glycosylated peptides. "variants" also include peptides which are modified after collection of a sample, for example, by covalent or non-covalent attachment of a label, especially radioactive, or should be labeled to the peptide.

Examples of specific variants and methods for their measurement are known (see, e.g., Ala-Kopsala, M., Magga, J., Peuhkurinen, K. et al (2004): molecular genetic has a major impact on the measurement of cyclic N-terminal fragments of A-type and B-type natural peptides. clinical chemistry, vol.50(9), 1576-1588).

Other preferred embodiments of the invention include combinations of different markers measured at the same time or at different times. An example is the measurement of the combination of NT-probnp and BNP.

In another preferred embodiment of the method of the invention, the amount of hemoglobin (Hb) in a sample of the patient will be determined. Hb can be determined by a variety of techniques well known to those skilled in the art. A significant increase in the amount of Hb would also indicate an increased risk of the need for dialysis to occur. A significantly reduced amount of Hb would indicate a reduced risk. The increased amount of Hb used according to the invention is preferably an amount above 11g/dL, more preferably an amount of 13 to 15g/dL, and the decreased amount is an amount below 11g/dL, more preferably below 10.5 g/dL.

Furthermore, also in a preferred method of the invention, previous medical history will be considered. In particular, it has been found in the studies of the present invention that previous cardiovascular diseases, preferably myocardial infarction, indicate an increased risk of developing a need for dialysis.

A decrease in creatinine clearance of at least 1ml/min is another indicator of an increased risk of the need for dialysis to occur. Thus, in another preferred embodiment of the invention, creatinine clearance as well as NT-proBNP may be determined. More preferably, creatinine clearance of 15 to 25mL/min indicates increased risk.

The term "diagnosing" as used herein refers to assessing the risk, i.e. probability, that a subject will develop a need for dialysis as described in the present specification. As will be understood by those skilled in the art, such an assessment is not generally intended to be correct for 100% of the subjects to be diagnosed. However, a statistically significant portion of subjects for which this term is required can be correctly diagnosed as developing a need for the dialysis. Whether a moiety is statistically significant can be immediately determined by one of skill in the art using a variety of well-known statistical evaluation tools, such as determining confidence intervals, p-value determination, student's t-test, Mann-Whitney test, and the like. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p value is preferably 0.05, 0.01, 0.005 or 0.0001. Preferably, the probabilities contemplated by the present invention allow a diagnosis to be correct for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population.

Diagnosis according to the present invention includes determining, monitoring, confident, sub-categorizing and predicting the relevant condition, risk or need. Determination involves awareness of a disease, risk, or need. Monitoring involves tracking the condition, risk or need that has been diagnosed, e.g., to analyze the progression of the disease or risk or the effect of a particular treatment on the progression of the disease or risk. It is believed to be involved in strengthening or proving a diagnosis that has been made using other indicators or markers. Sub-classification involves further defining the diagnosis according to different sub-categories of the condition, risk or need diagnosed, e.g., according to the mild and severe forms of the condition. Prediction involves diagnosing a condition, risk or need before other symptoms or markers become apparent or have become significantly altered.

As will be appreciated elsewhere herein, the risk stratification provided by the inventive method preferably involves a time window (prediction window) determined in the future. The prediction window is the time interval within which the subject will develop a need for dialysis according to the predicted probability. The prediction window may be the entire remaining life of the subject when analyzed by the method of the invention. Preferably, the prediction window is a time interval of several months up to two years after a sample to be analyzed by the method of the invention has been obtained. More preferably, the time window is disclosed in detail elsewhere in the specification.

The term "patient" according to the present invention relates to a healthy individual, an apparently healthy individual, or an individual, in particular, suffering from a disease. In particular, the patient suffers from a renal disorder, in particular chronic kidney disease (e.g. due to diabetic nephropathy), more particularly pre-terminal renal insufficiency or pre-terminal renal failure. Thus, the patient may have diabetes. Even more particularly, at the time of measurement or diagnosis, the patient does not require dialysis and/or does not require immediate dialysis already diagnosed.

The diagnosis according to the invention is preferably performed using a diagnostic tool. The diagnostic means are any means which allow to measure the level, amount or concentration of a substance of interest, in particular a peptide or polypeptide of interest, more in particular a BNP-type peptide.

Methods and diagnostic tools that can be used to measure the levels of the respective peptides are known to those skilled in the art. These include microplate ELISA-based methods, fully automated or robotic immunoassays (e.g., as in Elecsys^TMOr Cobas^TMAvailable on an analyzer), CBA (enzymatic cobalt binding assay, e.g.as can be found in Roche-Hitachi^TMOn the analyzerObtained) and latex agglutination assays (e.g.in Roche-Hitachi)^TMObtained on an analyzer). The method and means for measuring also include point-of-care devices, e.g. Cardiac Reader^TM(available from Roche Diagnostics).

A point-of-care device is generally understood to be a device that can be at the patient's bedside and that is capable of ambulatory testing and home care. An example is the Cardiac Reader^TM(available from Roche Diagnostics) in combination with, for example, NT-proBNP test strips (available as "Cardiac proBNP" from Roche Diagnostics). Such a test may use two (preferably monoclonal) antibodies against the peptide of interest (e.g., a BNP-type peptide). The antibodies can be used, for example, with Elecsys^TMOr Cobas^TMThe antibodies of the assay were identical. For example, a first antibody is labeled with biotin and a second antibody is labeled with gold particles. The test is initiated by adding a small volume (e.g., 150 μ L) of blood sample to the test strip (e.g., into the sample well of the test strip). For example, if the sample is flowed through a suitable fleece (e.g., a glass fiber fleece), red blood cells in the sample can be separated from the remaining plasma before or after addition to the test strip. The separation means (e.g. fleece) may preferably be part of a test strip. The antibody (preferably already present on the test strip) is dissolved in the remaining plasma. The antibodies are capable of binding to the peptide and polypeptide of interest to form a ternary sandwich complex. The antibody (bound or unbound) flows through the test strip into the monitoring zone. The monitoring band contains means for monitoring the bound complex, for example, it may contain streptavidin. This immobilizes the complex and the immobilized complex is displayed as a purple line by the gold-labeled antibody. Preferably, the remaining free gold-labelled antibody may move further down the band where it is captured in a region comprising a synthetic peptide or polypeptide comprising an epitope of the BNP-type peptide to be monitored and displayed as an isolated purple line. The presence of this second line can serve as a control as it indicates that the sample flow is working correctly and that the antibody is intact. The test strip may contain a marker indicating which peptide of interestOr the polypeptide can be detected using the test strip. It may also contain a bar code or other code graduated by the device for optically measuring the amount of detectable label in the detection zone. Such a barcode may include information indicating which peptide or polypeptide of interest can be detected using the test strip. The barcode may also include lot specific information about the test strip.

The Cardiac Reader itself contains a camera (e.g., a charge coupled device camera (CCD camera)) that optionally records the detection zone of the test strip. The signal and control lines can be identified by a pattern recognition algorithm. The intensity of the label in the signal line may be generally proportional to the amount of peptide or polypeptide of interest. The optical signal can be converted to concentration by a batch-specific calibration curve, which can be stored in a code chip. The calibration code and test lot identity can be checked by the bar code on the test strip.

Furthermore, the person skilled in the art is familiar with different methods for measuring the level of a peptide or polypeptide. The term "level" relates to the amount or concentration of a peptide or polypeptide in a patient or in a sample taken from a patient.

The term "measuring" according to the present invention relates to determining, preferably semi-quantitatively or quantitatively, the amount or concentration of a nucleic acid, peptide, polypeptide or other substance of interest. The measurement may be made directly or indirectly. Indirect measurements include measuring cellular responses, bound ligands, labels, or enzymatic reaction products. Preferably, the measurement is performed in vitro.

In the context of the present invention, an amount also relates to a concentration. Obviously, the concentration of the substance in question can be calculated from the total amount of the substance of interest in a sample of known size, and vice versa.

The measurement may be performed according to any method known in the art. Preferred methods are described below.

In a preferred embodiment, the method for measuring the level of a peptide or polypeptide of interest, in particular a BNP-type peptide, comprises the following steps: (a) contacting a cell capable of producing a cellular response to said peptide or polypeptide with said peptide or polypeptide for a sufficient time, (b) measuring said cellular response.

In another preferred embodiment, the method for measuring the level of a peptide or polypeptide of interest, in particular a BNP-type peptide, comprises the following steps: (a) contacting the peptide or polypeptide with a suitable substance for a sufficient time, (b) measuring the amount of product.

In another preferred embodiment, the method for measuring the level of a peptide or polypeptide of interest, in particular a BNP-type peptide, comprises the following steps: (a) contacting the peptide or polypeptide with a specifically binding ligand, (b) (optionally) removing unbound ligand, (c) measuring the amount of bound ligand.

Preferably, the peptides and polypeptides are contained in a sample, in particular a body fluid or tissue sample, and the amount of the peptides or polypeptides in the sample is measured.

Peptides and polypeptides (proteins) can be measured in tissue, cell and body fluid samples, i.e. preferably in vitro. Preferably, the peptide or polypeptide of interest is measured in a sample of bodily fluid.

A tissue sample according to the invention refers to any type of tissue obtained from a dead or living human or animal body. The tissue sample may be obtained by any method known to those skilled in the art, for example, by biopsy or curettage.

The term "body fluid sample" according to the present invention preferably relates to a blood sample or a derivative thereof. More preferably, the term relates to plasma or serum. The body fluid sample may be obtained by any known and deemed suitable method.

Methods of obtaining a cell sample include directly preparing individual cells or groups of small cells, dissociating tissue (e.g., using trypsin), and separating cells from body fluids, e.g., by filtration or centrifugation. The cells according to the invention also comprise platelets and other anucleated cells, for example, erythrocytes.

The sample may be further processed if necessary. In particular, nucleic acids, peptides or polypeptides may be purified from a sample according to methods known in the art, including filtration, centrifugation, live extraction methods, such as chloroform/phenol extraction.

To measure cellular responses, the sample or treated sample is added to a cell culture and internal or external cellular responses are measured. The cellular response may include expression of a reporter gene or secretion of a substance such as a peptide, polypeptide, or small molecule.

Other preferred methods of measurement include measuring the amount of ligand that specifically binds to the peptide or polypeptide of interest. Binding according to the present invention includes both covalent and non-covalent binding.

The ligand according to the invention may be any one of the peptides or polypeptides of interest, polypeptides, nucleic acids or other substances that bind. It is well known that peptides or polypeptides are obtained from human or animal cells and may then be modified, for example by glycosylation. Suitable ligands according to the invention may also bind to the peptide or polypeptide through such sites.

Preferably, the ligand should specifically bind to the peptide or polypeptide to be measured. According to the present invention, "specifically binds" means that the ligand should not substantially bind (be "cross-reactive" with) another peptide, polypeptide or substance present in the sample under investigation. Preferably, the specifically bound protein or isoform should bind with at least 3-fold, more preferably at least 10-fold, even more preferably at least 50-fold higher affinity than any other related peptide or polypeptide. In this context, such other related peptides or polypeptides may be other structurally related or homologous peptides or polypeptides.

Non-specific binding may be tolerable, especially if the peptide or polypeptide of interest can still be clearly distinguished and measured, for example on a western blot depending on its size or by its relatively high abundance in the sample.

Binding of the ligand can be measured by any method known in the art. Preferably, the method is semi-quantitative or quantitative. Suitable methods are described below.

First, the binding of the ligand can be measured directly, such as by NMR or surface plasmon resonance.

Secondly, if the ligand also serves as a substrate for the enzymatic activity of the peptide or polypeptide of interest, the enzymatic reaction product can be measured (e.g., the amount of protease can be measured by measuring the amount of substrate cleaved, e.g., on a western blot).

For the measurement of the enzymatic reaction product, it is preferred that the amount of substrate is saturated. The substrate may also be labeled with a detectable label prior to the reaction. Preferably, the sample is contacted with the substrate for a sufficient time. Sufficient time refers to the time necessary to produce a detectable, preferably measured, amount of product. Instead of measuring the amount of product, the time required for a given (e.g., detectable) amount of product to appear can be measured.

Third, the ligand may be coupled covalently or non-covalently to a label, allowing detection and measurement of the ligand.

Labeling may be performed by direct or indirect methods. Direct labeling involves coupling the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves the binding of a secondary ligand (covalent or non-covalent) to a primary ligand. The secondary ligand should specifically bind to the primary ligand. The secondary ligand may be coupled to a suitable label and/or may be the target (receptor) of a tertiary ligand that binds the secondary ligand. The use of secondary, tertiary or even higher order ligands is often used to enhance the signal. Suitable second or higher order or ligands may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.).

One or more labels known in the art may also be used to "label" the ligand or substrate. Such labels may then be targets for higher order ligands. Suitable tags include biotin, digoxin, His tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus Hemagglutinin (HA), maltose binding protein, and the like. In the case of peptides or polypeptides, the label is preferably at the N-terminus and/or C-terminus.

Suitable labels are any labels that can be detected by a suitable detection method. Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels (e.g., "magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels.

Enzymatically active labels include, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, and derivatives thereof. Suitable substrates for detection include bis-amino-benzidine (DAB), 3 '-5, 5' -tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as a ready stock solution from Roche Diagnostics), CDP-Star^TM(Amersham Biosciences)、ECF^TM(Amersham Biosciences). Suitable enzyme-substrate combinations may result in colored reaction products, fluorescence or chemiluminescence, which may be measured according to methods known in the art (e.g., using a light-sensitive film or a suitable camera system). With regard to measuring the enzymatic reaction, the criteria given above can be applied analogously.

Typical fluorescent labels include fluorescent proteins (such as fluorescent proteins from jellyfish victoria multitubular luminescent jellyfish (such as GFP, YFP, RFP and derivatives thereof) or fluorescent proteins of pansy renilla), Cy3, Cy5, texas red, luciferin, Alexa dyes (e.g., Alexa568), and quantum dots. Other fluorescent labels may be obtained, for example, from Molecular Probes (Oregon).

Exemplary radiolabels include³⁵S、¹²⁵I、³²P、³³P、³H, and the like. The radiolabel may be detected by any known and suitable method, such as a light-sensitive film or a phosphoimager.

Suitable measurement methods according to the invention also include precipitation (especially immunoprecipitation), electrochemiluminescence (electrogenic chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immunoassay, electrochemiluminescence, sandwich immunoassay (ECLIA), dissociation-amplified lanthanide fluorescence immunoassay (DELFIA), Scintillation Proximity Assay (SPA), turbidimetry, nephelometry, latex-enhanced turbidimetry or nephelometry, solid phase immunoassay, and mass spectrometry, such as SELDI-TOF, MALDI-TOF or capillary electrophoresis mass spectrometry (CE-MS). Other methods known in the art (e.g., gel electrophoresis, two-dimensional gel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting) can be used alone or in combination with labeling or other detection methods as described above.

Preferred ligands include antibodies, nucleic acids, peptides or polypeptides, and aptamers, such as nucleic acid or peptide aptamers (e.g., spiegelmers or anti-carrier proteins). Methods for obtaining such ligands are known in the art. For example, the supplier also provides for the identification and production of suitable antibodies or aptamers. The person skilled in the art is familiar with methods for developing derivatives of such ligands with higher affinity or specificity. For example, random mutations can be introduced in a nucleic acid, peptide, or polypeptide. Binding of these derivatives can be tested according to screening methods known in the art, such as phage display.

The term "antibody" as used herein includes polyclonal and monoclonal antibodies, as well as variants or fragments thereof, such as Fv, Fab and F (ab) capable of binding an antigen or hapten₂And (3) fragment. The term "antibody" also includes single chain antibodies.

In another preferred embodiment, a ligand, preferably selected from a nucleic acid, a peptide, a polypeptide or an aptamer, is present on the array.

The array contains at least one additional ligand, which may be directed against a peptide, polypeptide or nucleic acid of interest. The additional ligands may also be directed against a peptide, polypeptide or nucleic acid of a non-specific purpose in the context of the present invention. Preferably, ligands of at least three, preferably at least five, more preferably at least eight peptides or polypeptides of interest in the context of the present invention are comprised on the array.

According to the present invention, the term "array" refers to a solid phase or gel-like support on which at least two compounds are attached or bound in a one-, two-or three-dimensional arrangement. Such arrays (including "gene chips", "protein chips", antibody arrays, etc.) are well known to those skilled in the art and are typically produced on glass slides, particularly coated glass slides such as polycation-, cellulose-or biotin-coated glass slides, coverslips, and membranes, such as nitrocellulose-or nylon-based membranes.

The array may comprise bound ligands or at least two cells, each cell expressing at least one ligand.

In another preferred embodiment, the ligand, preferably selected from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, is present on a solid support, preferably on an array. According to the present invention, the term "array" ("gene chip", "protein chip", antibody array, etc.) refers to a solid phase or gel-like support on which at least two compounds are attached or bound in a one-, two-or three-dimensional arrangement. Solid supports or arrays comprising ligands or binders to BNP-like peptides are well known in the art and include commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction wells, plastic tubes, and the like. The ligand or binding agent may be bound to a number of different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agarose, and magnetite. For the purposes of the present invention, the nature of the carrier may be soluble or insoluble. Suitable methods for immobilizing/immobilizing the ligand or binding agent are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like.

It is also envisaged to use "Suspension arrays" as arrays according to the invention (Nolan JP, Sklar LA. (2002); Suspension array technology: evolution of the flat-array partner. trends Biotechnology.20 (1): 9-12). In such suspension arrays, carriers, such as microbeads or microspheres, are present in a suspension. The array consists of different microbeads or microspheres, possibly labeled, carrying different ligands.

The invention also relates to a method of producing an array as defined above, wherein at least one ligand and further ligands are bound to a support material.

Methods for producing such supports, for example, based on solid phase chemistry and photolabile protecting groups are well known (US 5,744,305). Such arrays may also be contacted with a substance or library of substances and tested for interaction, e.g., for binding or confirmed change. Thus, an array comprising a peptide or polypeptide as defined above may be used to identify ligands that specifically bind the peptide or polypeptide.

The invention thus also relates to the use of a diagnostic tool capable of measuring, preferably in vitro, the level of a BNP-type peptide, in particular NT-probnp, in a patient, for diagnosing the risk of developing a need for dialysis.

The invention also relates to a kit comprising means or agents for measuring BNP-type peptides. Such means or agents may be any suitable means or agents known to those skilled in the art. Examples of such tools or agents and methods of their use have been given in the specification. For example, a suitable agent may be any type of ligand or antibody capable of specifically binding a BNP-type peptide. The kit may further comprise any other components deemed suitable in the context of measuring the level of the respective biomarker, such as suitable buffers, filters, etc.

Optionally, the kit may additionally comprise a user manual for interpreting the results of any measurement regarding the risk of diagnosing the patient's need for developing dialysis. In particular, such a manual may include information about which measured level corresponds to which level of risk. This is outlined in detail elsewhere in this specification. Furthermore, such user manual may provide guidance on the correct use of the components of the kit for measuring the levels of the respective biomarkers.

The invention also relates to the use of said kit for diagnosing the risk of developing a need for dialysis. The invention also relates to the use of said kit in any of the methods according to the invention for diagnosing the risk of developing a need for dialysis.

Furthermore, the invention comprises a device for diagnosing the risk of developing a need for dialysis, comprising:

a) means for measuring the amount of a BNP-type peptide in a sample from a patient; and

b) means for diagnosing said risk by comparing the measured level with at least one reference level.

The invention also relates to the use of such a device for diagnosing the risk of developing a need for dialysis.

The term "device" as used herein relates to a tool system comprising at least one of the above-mentioned tools operatively connected to each other so as to allow diagnosing the risk of developing a need for dialysis. Preferred means for measuring the level of BNP-type peptides and diagnosing said risk are disclosed elsewhere in this specification in relation to the methods of the invention. How the tool is operatively connected will depend on the type of tool included in the device. For example, when a tool for automatically measuring the level of a BNP-type peptide is applied, the data obtained by means of said automated operating tool can be processed, for example by means of a computer program, in order to diagnose said risk. Preferably, in this case, the tool is contained in one device. The device may thus comprise an analysis unit for measuring the level of the BNP-type peptide in the applied sample and a computer unit for processing the resulting data for diagnosis. Alternatively, when the level of the BNP-type peptide is measured with a tool such as a test strip, the diagnostic tool may comprise a control test strip or table which assigns the measured level to a reference level as defined elsewhere in the specification. The test strip is preferably coupled to a ligand or agent that specifically binds to the BNP-type peptide. The test strip or device preferably comprises means for detecting binding of a BNP-type peptide to the ligand or agent. Preferred detection means are disclosed in the embodiments relating to the inventive methods described above. In this case, the means are operatively connected in that the user of the system puts together the results of the determination of the quantity and its diagnostic value according to the instructions and explanations given in the manual. The means may be present in such an embodiment as a separate device and preferably packaged as a kit. Those skilled in the art will recognize how to attach the tool without taking a dilemma. Preferred devices are those that can be used without the expertise of a clinician, such as test strips, or electronic devices that merely require a sample to be loaded. The results may be output as diagnostic parameters or raw data that needs to be interpreted by a clinician. Other preferred devices include analytical units/devices (e.g. biosensors, arrays, solid supports coupled to specifically recognize BNP, plasmon surface resonance devices, NMR spectrometers, mass spectrometers, etc.) or evaluation units/devices as referred to above according to the method of the invention.

The method according to the invention comprises a step of diagnosing the risk of the patient, which comprises comparing the level of the BNP-type peptide with at least one reference level, e.g. a known level associated with a different level of risk in the patient.

According to the present invention, the term "risk" relates to the probability that a specific event, in particular the need for dialysis, occurs. For example, a risk may be that a given event will occur with at least a 2%, 5%, 10%, 15%, or 20% probability in an individual patient within a particular time frame. Preferably, the predicted time range according to the present invention is at least 1 month, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months or up to 24 months. Clinical studies may provide data indicative of such risk. From the above, it is clear that the risk diagnosis will also allow for a prediction of the time interval within which or until then the need for dialysis does not occur for the patient. Generally, the higher the risk, the shorter the time interval will be. It will also predictively determine the probability that a patient will develop a need for dialysis within a given time interval.

The given risk may be derived from, for example, a Kaplan-Meier curve of time versus a given event. See, for example, example 3 and the specific risks and hazard ratios mentioned therein.

Although risk may be expressed in absolute values, it is often more useful to express the risk in relative terms ("relative risk"), for example, as an increased or highly increased risk relative to a particular given risk or relative to a control group in a clinical study, a patient at another stage of renal failure, or even an age-matched normally healthy person. Those skilled in the art are well familiar with such relative terms. For example, in the general population, renal patients, or patients with a particular renal disease, there is an average given risk of developing a need for dialysis. However, it may be more relevant to know the additional risk of a particular patient developing a need for dialysis compared to the respective comparison group (e.g. the mentioned general population, renal patients or patients suffering from a particular renal disease), so that the overall risk of the patient is "increased". Advantageously, the present invention also allows diagnosing such relative risks.

The relative risk can be expressed in a hazard ratio. The term "hazard ratio" is known to those skilled in the art. It may express a relationship of risk between two subgroups, e.g. a hazard ratio between a group with a low level of a BNP-type peptide and a group with that level of a BNP-type peptide. Differences in hazard ratios are known, for example, as interactions that would be extracted from interaction models of risk groups with certain BNP levels. The terms interaction and interaction model are known to those skilled in the art.

In particular, the invention allows to identify patients at a certain risk of developing a need for dialysis. For example, the risk may be increased, not increased, or decreased. Those skilled in the art are familiar with the meaning of these terms. For example, if a particular patient has a higher risk than the average patient, the technician will often refer to such risk as "increased". Preferably, the term "increased risk" is understood as that the patient is more likely to develop a need for dialysis or that the patient will develop a need for dialysis earlier than a typical comparable patient. Preferably, patients with increased risk should be cared for, which focuses on the development of a need for dialysis. Those skilled in the art will appreciate that the ultimate decision on treatment will be made by the responsible physician, who will consider additional relevant factors such as the age of the patient, the family history of renal disease, the nature or cause of renal disorders present in the patient, available treatment options, the availability of monitoring possibilities, and the like.

In the context of the present invention, the increased risk of developing a need for dialysis especially relates to an increased risk of at least 1.5-fold, 2-fold, 3-fold, 3.5-fold or 4-fold compared to an ordinary patient, preferably compared to an ordinary patient of the same age and sex, more preferably compared to the same age, sex and nature or cause of the renal disorder of the patient.

The person skilled in the art is able to determine the known levels of BNP-type peptides associated with different levels of risk of developing a need for dialysis.

According to the invention, the higher the measured level of BNP-type peptide, the higher the risk of developing a need for dialysis.

Preferably, the risk is determined by comparing the measured level of the BNP-type peptide with a reference level. The term "reference level" is known to the person skilled in the art. In particular, the reference level may be associated with a specific risk or it may distinguish between different levels of risk. It will be appreciated that the reference level may also be selected according to the desired diagnostic sensitivity or specificity. Higher sensitivity means that a larger fraction of all patients with a particular diagnosis are identified and/or fewer patients with a particular diagnosis are incorrectly diagnosed as having the diagnosed disease, complication or risk. Higher specificity means that a larger fraction of patients identified as having a particular diagnosis actually have the disease, complication, or risk diagnosed. The higher the desired sensitivity of a particular diagnosis, the lower the specificity of the diagnosis and vice versa. Thus, the reference level can be selected by one skilled in the art based on the desired sensitivity and specificity.

In the context of this discussion, it is clear that a reference level may not be merely a single value, but may also include a range of values.

More specifically, the reference level may be derived from the level of the BNP-type peptide determined in a clinical study, for example as given in the examples.

An example of reference levels is given below, i.e. plasma levels of NT-probnp are given, which have been found in the course of the present invention to be related to or to distinguish between said levels of risk of developing a need for dialysis.

Obviously, the levels given below may only serve as a first classification of patient risk. For example, the risk may also depend on the general physical state of the patient and the nature of the underlying condition that poses a risk of suspected development of a need for dialysis.

According to the invention, for example, the level corresponding to a plasma level of less than 500pg/mL, more particularly less than 400pg/mL, most particularly less than 300pg/mL of NT-probnp is associated with no increased risk of developing a need for dialysis.

According to the invention, for example, a level equal to or less than 300pg/mL, more preferably equal to or less than 400pg/mL, most particularly equal to or less than 500pg/mL of NT-probnp is associated with an increased risk of developing a need for dialysis.

Clearly, the given levels may overlap depending on the sensitivity and specificity chosen. Thus, according to the invention, for example, the levels corresponding to plasma levels of 300 to 500pg/mL, more particularly 350 to 450pg/mL, more particularly 380 to 420pg/mL, in particular 400pg/mL NT-probnp are able to distinguish between a non-increased risk and an increased risk of developing a need for dialysis. If the measured level is higher than the discrimination level, the measured level indicates an increased risk of developing a need for dialysis. Such a level of discrimination may also be referred to as a "cut-off value" or "decision threshold". Such a "cut-off value" or "decision threshold" may tell the responsible physician whether to perform a routine treatment as planned or to initiate a treatment or monitoring taking into account the increased risk of dialysis occurring.

Once the risk in the patient has been diagnosed, the following subsequent treatment may be performed accordingly. The risk levels mentioned below refer in particular to the risk levels associated with the above-mentioned NT-proBNP levels.

If the method according to the invention indicates an increased risk, the treatment can be performed as planned. ESA treatment may be accompanied by monitoring NT-probnp levels at loose event intervals, such as every 4 weeks, 2 months or 3 months.

If the method according to the invention indicates an increased risk, treatment may be performed. Preferably, treatment will be accompanied by further measurement of the level of BNP-type peptides of the invention and further diagnosis, such as monitoring of renal function at relatively close intervals, for example about monthly, preferably about biweekly, or about weekly. Thus, the present invention also provides a method of treating or monitoring a patient at risk of developing a need for dialysis.

The invention also relates to monitoring the risk of developing a need for dialysis. Furthermore, the invention relates to further monitoring the risk once it has been diagnosed.

The term "monitoring" is known to the person skilled in the art and has been defined elsewhere in the present application. "closer monitoring" preferably involves monitoring at shorter event intervals than in a typical patient. For example, closer monitoring can be performed by diagnosing risk in a patient at regular intervals, such as at intervals of about 12 hours, 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 4 months, 6 months, or 1 year.

The term "about" in this context is understood by those skilled in the art. Thus, the actual interval may deviate from the expected regular interval, depending on the actual circumstances, such as the arrangement of appropriate reservations, etc. In particular, the time interval may for example deviate by at most 100%, preferably at most 50%, more preferably at most 25%, most preferably at most 10%.

An additional advantage of monitoring is to observe whether a certain treatment is successful in reducing the need for dialysis to develop and/or delaying the need for dialysis.

Any preferred embodiments or features mentioned in this specification apply of course correspondingly in the monitoring aspect.

In another embodiment, the invention relates to a method of determining the risk of more closely monitoring the need for developing dialysis, in particular in renal disorder patients, comprising the steps of: (a) measuring, preferably in vitro, the level of the BNP-type peptide, (b) diagnosing the risk of the patient developing the need for dialysis by comparing the level of the BNP-type peptide with known levels associated with different levels of risk in the patient, (c) recommending that closer monitoring be initiated or prevented. Preferably, closer monitoring is recommended if the method indicates an increased risk of developing a need for dialysis. It is clear that the method can be varied according to all embodiments or preferred aspects of the invention mentioned in the present description.

It is clear that all the tools and methods provided herein can be advantageously combined with tools and methods deemed suitable by the skilled person, e.g. risk diagnosis or monitoring according to the present invention can be accompanied by measuring glomerular filtration rate or determining changes in glomerular filtration rate.

Finally, the invention also encompasses the use of a device or a BNP-type peptide or variant thereof as described herein for diagnosing the risk of developing a need for dialysis.

All references cited in this specification are incorporated herein by reference for their full functional content and the disclosure specifically mentioned in this specification.

The figure shows a Kaplan-Meier graph comparing treatment groups based on endpoint "need for dialysis", a) time versus dialysis Kaplan-Meier curves showing the disadvantages of group 1 (faster than dialysis), B) dialysis events for baseline NT-probnp showing a higher probability that baseline NT-probnp results in a higher "need for dialysis".

The following examples illustrate the invention and are not intended to limit the invention in any way.

Example 1: measurement of NT-proBNP

NT-proBNP was determined by electrochemiluminescence immunoassay (ELECSYS ProBNP sandwich immunoassay; Roche Diagnostics, Mannheim, Germany) on ELECSYS 2010. The assay operates according to the principle of electrochemiluminescence sandwich immunoassay. In the first step, a biotin-labeled IgG (1-21) capture antibody, ruthenium-labeled F (ab')₂(39-50) Signal antibody and 20. mu.l sample were incubated at 37 ℃ for 9 minutes. Afterwards, streptavidin-coated magnetic microparticles were added and the mixture was incubated for a further 9 minutes. After the second incubation, the reaction mixture is transferred to the measurement chamber of the system, where the beads are magnetically captured to the electrode surface. Unbound label is removed by washing the measurement chamber with buffer.

In the final step, a voltage is applied to the electrodes in the presence of a tripropylamine-containing buffer and the electrochemiluminescence signal is recorded by a photomultiplier tube. By passingThe device is fully automated to handle all reagents and samples. The results were determined by a calibration curve generated in an instrument-specific manner by a two-point calibration method and the main curve was provided by the reagent barcode. The tests were performed according to the manufacturer's instructions.

Example 2: obtaining of samples

Blood samples for BNP-type peptide analysis were collected, if appropriate, using EDTA-tubes or lithium-heparin tubes (for clinical chemistry) containing 5000U of aprotinin (Trasylol, Beyer, Germany). Blood and urine samples were immediately centrifuged at 3400rpm for 10 minutes at 4 ℃. The supernatant was stored at-80 ℃ until analysis.

Example 3: NT-proBNP is a marker for the need for dialysis

The CREATE study is an open, randomized, parallel-group, multicenter study to study the effect of early anemia correction with betaepoetin on reducing cardiovascular risk in patients with chronic renal anemia who are not undergoing renal replacement therapy. The primary objective of this study was to study the effect of early betaepoetin treatment to target hemoglobin (Hb) levels of 13-15g/dL on cardiovascular morbidity and compare these effects to that obtained with betaepoetin treatment to maintain target Hb levels of 10.5-11.5 g/dL. The primary endpoint is the combined endpoint (time to first event) of all regimen-specific cardiovascular events: prolonged angina resulting in hospitalization for at least 24 hours or hospitalization, acute heart failure, fatal or non-fatal myocardial infarction, fatal or non-lethal stroke, sudden death, Transient Ischemic Attack (TIA), peripheral vascular disease (amputation, necrosis), prolonged arrhythmia resulting in hospitalization for at least 24 hours or hospitalization. In other events, the need for dialysis was studied as a secondary endpoint for the patient.

Additional laboratory measurements were performed at baseline, 6, 12, 24, 36, and 48 months in a subset of patients with CREATE in the NT-probnp collateral study. The measurements performed were NT-probnp and some other laboratory measurements.

The entire study population, including patients from the NT-probnp-adjunct study, was followed for the occurrence of events eligible for the predetermined study, with the first study endpoint and all second study endpoints including events to dialysis. All individual events, including the need for dialysis, were classified by the respective independent end point committee or professional Roche research staff according to the Roche standard procedures (SOPs). The need to initiate renal replacement therapy, such as dialysis or renal transplantation, was collected and evaluated for each patient on a specially designed page of the medical record report form. The respective data are evaluated according to a predetermined statistical analysis plan.

In the first preliminary analysis, the baseline NT-probnp levels of 226 patients in the CREATE study population were divided into 2 treatment groups ("Hb high" being a Hb target value of 13 to 15g/dL and "Hb low" being a Hb target value of 10.5 to 11.5 pg/dL). In addition, these groups were separated by preliminary median NT-probnp levels (> 400 and < 400pg/mL) for the entire population. The time to dialysis was plotted in the Kaplan Meier plot of fig. 1 as defined by the end point of the study (e.g., the fraction of the population that has not experienced this particular event). Hazard ratios (i.e., factors by which the respective risks increase or decrease) were calculated and p-values < 0.05 were considered significant. A Kaplan-Meier plot of events leading to the development of the need for dialysis was generated (fig. 1). An overview of the incidence (time to ignore events) of the two treatment groups is provided by table 1 below.

Table 1:

patients with values above 400pg/mL developed a need for dialysis statistically significantly more often in both treatment strategy groups than patients with values below 400pg/mL, the "Hb high" group having a higher risk ratio.

In table 2 below, the risk factors for developing a need for dialysis are given as well as the corresponding p-values of various parameters including NT-probnp levels, Hb levels and creatinine clearance, as determined based on the results of the above-mentioned study.

Table 2:

Claims (12)

1. A method for diagnosing the need to develop dialysis in a patient, comprising the steps of:

a) measuring the level of a BNP-type peptide or variant thereof in a sample from the patient,

b) diagnosing said risk by comparing the measured level of the BNP-type peptide or variant thereof with at least one reference level.

2. The method of claim 1, wherein the patient has a renal condition.

3. A method according to any one of claims 1 to 3, wherein the BNP-type peptide is BNP, probnp, NT-probnp or a variant thereof.

4. The method according to claim 3, wherein the BNP-type peptide is NT-proBNP or a variant thereof.

5. The method according to any one of claims 1 to 4, wherein the reference level corresponds to a plasma level of NT-proBNP of 300 to 500pg/mL, in particular 350 to 450 pg/mL.

6. A method according to any one of claims 1 to 5, wherein a measured level above said reference level is indicative for an increased said risk.

7. Method according to claim 6, wherein said increased risk relates to an increase of at least 1.5 fold, in particular 2 fold, more in particular 3 fold, compared to the risk of a general patient, preferably of the same age, sex and general patient suffering from a condition causing renal insufficiency.

8. The method according to any one of claims 1 to 7, wherein the level of the BNP-type peptide is measured using a specific binding ligand, preferably an antibody or an aptamer.

9. Use of a diagnostic tool capable of measuring the level of a BNP-type peptide, in particular NT-probnp, for diagnosing the risk of developing a need for dialysis.

10. Use of a kit comprising means capable of measuring the level of a BNP-type peptide or a variant thereof, in particular NT-probnp or a variant thereof, for diagnosing the risk of developing a need for dialysis.

11. Device for diagnosing the risk of developing a need for dialysis in a patient, comprising

(a) Means for measuring the amount of a BNP-type peptide or variant thereof in a sample from a patient; and

(b) means for diagnosing said risk by comparing the measured level with at least one reference level.

12. A method for determining a more closely monitored risk of developing a need for dialysis, comprising the steps of:

(a) measuring, preferably in vitro, the level of a BNP-type peptide,

(b) diagnosing the risk of the patient to develop a need for dialysis by comparing the measured level of the BNP-type peptide with known levels associated with different levels of risk in the patient,

(c) it is recommended to initiate a closer monitoring or to avoid a closer monitoring.