Markers for statin therapy stratification in heart failure
阅读说明：本技术 在心力衰竭中用于抑制素治疗分层的标记物 (Markers for statin therapy stratification in heart failure ) 是由 D.布洛克 H.布伦纳 T.迪特勒 U-H.温许斯-特伦 C.曹格 A.齐格勒 于 2014-08-26 设计创作，主要内容包括：本发明涉及在心力衰竭中用于抑制素治疗分层的标记物。本发明涉及鉴定具有心力衰竭的患者为可能对包括抑制素的治疗应答的方法。所述方法基于在患者的样品中测量选自以下的至少一种标记物的水平：GDF-15(生长分化因子15)、尿素、SHBG(性激素结合球蛋白)、尿酸、PLGF(胎盘生长因子)、IL-6(白介素-6)、转铁蛋白、心肌肌钙蛋白、sFlt-1(可溶性fms-样酪氨酸激酶-1)、前白蛋白、铁蛋白、骨桥蛋白、sST2(可溶性ST2)和hsCRP(高敏C-反应蛋白)。进一步设想的是预测患者遭受死亡或住院治疗的风险的方法,其中所述患者具有心力衰竭且经历包括抑制素的治疗。所述方法还基于测量上文提及的标记物中至少一种的水平。(The present invention relates to markers for statin therapy stratification in heart failure. The present invention relates to methods of identifying patients with heart failure as likely to respond to treatment comprising statins. The method is based on measuring in a sample of the patient the level of at least one marker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive C-reactive protein). Further envisaged is a method of predicting the risk of a patient suffering from death or hospitalization, wherein said patient has heart failure and is undergoing a treatment comprising statin. The method is also based on measuring the level of at least one of the markers mentioned above.)
1. A method of identifying a patient with heart failure as likely to respond to a treatment comprising a statin, comprising:
(a) measuring the level of the biomarker sFlt-1 (soluble fms-like tyrosine kinase-1) in a sample of a patient, and
(b) sFlt-1 levels were compared to reference levels.
2. The method according to claim 1, wherein a level of the biomarker in the patient sample below the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample above the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
3. A method of identifying a patient with heart failure as likely to respond to a treatment comprising a statin, comprising:
(a) measuring in a patient sample the level of at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), SHBG (sex hormone binding globulin), IL-6 (interleukin-6), Urea, transferrin, cardiac troponin, prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive C-reactive protein), and
(b) comparing the level of the at least one marker to a respective reference level.
4. The method according to any one of claims 1 to 3, wherein the subject has heart failure classified according to the ACC/AHA classification as stage B, C or D, in particular as stage B or C according to the ACC/AHA classification, and/or wherein the subject has heart failure classified as NYHA type II, III or IV, in particular as NYHA type II or III according to the NYHA classification.
5. The method according to claim 3 or 4, wherein the patient further has coronary artery disease, in particular wherein the at least one biomarker is transferrin, ferritin, urea or IL-6.
6. The method of claim 3 or 4, wherein the patient does not have coronary artery disease, in particular wherein the biomarker is osteopontin or sST 2.
7. The method of any one of claims 1-6, wherein the statin is selected from the group consisting of: atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin.
8. The method of any one of claims 1-7, wherein the patient has been treated with statin prior to obtaining the sample.
9. The method of any one of claims 1-8, wherein the patient has not been treated with statin prior to obtaining the sample.
10. The method of any one of claims 3-9, wherein
i) At least one biomarker is GDF-15, and
a) wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is lower than a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
ii) at least one biomarker is SHBG, and
a) wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient sample that is lower than a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than a reference level indicates that the patient is less likely to respond to a treatment comprising statin, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
iii) the at least one biomarker is IL-6, and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
iv) the at least one biomarker is urea, and wherein a level of the biomarker in the patient sample above a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample below a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
v) the at least one biomarker is osteopontin, and wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient's sample below a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient's sample above a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
vi) the at least one biomarker is sST2, and wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient's sample that is below the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient's sample that is above the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
vii) the at least one biomarker is transferrin, and wherein the patient also suffers from coronary artery disease, and wherein a level of the biomarker in the patient's sample above a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient's sample below a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
viii) at least one biomarker is ferritin, and wherein the patient further suffers from coronary artery disease, and wherein a level of the biomarker in the patient's sample below a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient's sample above a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
ix) at least one biomarker is cardiac troponin, and
a) wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient sample that is lower than a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than a reference level indicates that the patient is less likely to respond to a treatment comprising statin, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
and/or
x) wherein at least one biomarker is prealbumin and/or hsCRP, and
a) wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
11. The method of any one of claims 1-10, wherein the sample is a blood, serum, or plasma sample.
12. The method of any one of claims 1-11, wherein the patient is a human.
13. In a sample of a patient with heart failure: i) at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (high sensitive c-reactive protein) and/or ii) at least one binding agent that specifically binds a biomarker selected from the group consisting of: use of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (high sensitive c-reactive protein) for identifying a patient as likely to respond to a treatment comprising a statin.
14. Apparatus for carrying out the method according to any one of claims 1 to 12, the apparatus comprising
a) An analyzer unit comprising at least one binding agent that specifically binds to at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitivity c-reactive protein), said unit being adapted for measuring the level of one or more biomarkers in a sample of a patient with heart failure, and
b) an analyzer unit for comparing the determined level with a reference level, whereby the patient is identified as more or less likely to respond to a treatment comprising inhibin, said unit comprising a database with one (or more) reference levels and a computer-implemented diagnostic algorithm for carrying out the comparison, in particular wherein said diagnostic algorithm is an algorithm as claimed in claim 2 or 10.
15. Inhibin for use in the treatment of heart failure in a patient having such a level of at least one biomarker, in particular blood, serum or plasma level, selected from the group consisting of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (hypersensitive c-reactive protein), which level is higher or lower than a respective reference level, in particular wherein
i) At least one biomarker is GDF-15, and
a) wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is higher than a reference level, or
b) Wherein the patient has coronary artery disease, and wherein the level of the biomarker is lower than a reference level,
ii) at least one biomarker is SHBG, and
a) wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is lower than a reference level, or
b) Wherein the patient has coronary artery disease, and wherein the level of the biomarker is higher than a reference level,
iii) at least one biomarker is IL-6, and wherein the level of said biomarker is higher than a reference level,
iv) at least one biomarker is urea, and wherein the level of said biomarker is higher than a reference level,
v) at least one biomarker is osteopontin, and wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is lower than a reference level,
vi) at least one biomarker is sST2, and wherein the patient does not have coronary artery disease, and wherein the level of the biomarker is lower than a reference level,
vii) at least one biomarker is sFlt-1, and wherein the level of said biomarker is lower than a reference level,
viii) at least one biomarker is transferrin, and wherein the patient also suffers from coronary artery disease, and wherein the level of the biomarker is higher than a reference level,
ix) at least one biomarker is ferritin, and wherein the patient also suffers from coronary artery disease, and wherein the level of said biomarker is lower than a reference level,
x) at least one biomarker is cardiac troponin, and
a) wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is lower than a reference level, or
b) Wherein the patient has coronary artery disease, and wherein the level of the biomarker is higher than a reference level,
and/or
xi) at least one biomarker is prealbumin and/or hsCRP, and
a) wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is higher than a reference level, or
b) Wherein the patient has coronary artery disease, and wherein the level of the biomarker is lower than a reference level.
Technical Field
The present invention relates to methods of identifying patients with heart failure as likely to respond to treatment comprising statins. The method is based on measuring in a sample of the patient the level of at least one marker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive C-reactive protein). A further concept is a method of predicting the risk of a patient suffering from death or hospitalization, wherein said patient has heart failure and is on a treatment comprising statin. The method is also based on measuring the level of at least one of the markers mentioned above.
Background
Heart Failure (HF) is a leading cause of morbidity and mortality in many countries worldwide. With the exception of Ivabradine (Ivabradine) and Cardiac Resynchronization Therapy (CRT), there has been no new treatment for heart failure in recent years. In contrast, most drug candidates over the last decade have failed at or before development stage III.
At the same time, statin therapy has become the fundamental treatment in the primary and secondary prevention of Coronary Artery Disease (CAD). Although CAD underlies many heart failure cases, treatment with statins in Chronic Heart Failure (CHF) is not supported by major guidelines. This is because inhibin does not function in the large scale phase III assay of CHF (neutral). In particular, the CORONA and GISSI-HF trials prospectively investigated the use of 10 mg daily rosuvastatin in patients with CHF (J Am Coll Cardiol 2009; 54: 1850-9; Lancet 2008; 372: 1231-9). Neither test demonstrated the beneficial effects of statin therapy at their initial endpoint.
The results of the CORONA and GISSI-HF trials led to the exclusion of statin treatment in the HF guidelines. However, there may be a subset of HF patients that may benefit from statin therapy. The causal analysis of the Heart Protection Study (Lancet. 2002; 360: 7-22) and the CORONA assay demonstrated the reduced benefit of statin treatment in patients with higher NT-proBNP levels. Similarly, statins are less beneficial in patients with high galectin-3 levels (Eur Heart j.2012; 33: 2290-6). However, this finding about the benefits of galectin-3 and inhibin may not be confirmed in the analysis of the GISSI-HF study (Latini r., personal communication).
Bonaca et al 2011 (Arterioscler Thromb Vase biol. 2011 Jan;31(1):203-10) discloses studies in which GDF-15 was analyzed in a discharged subject as a marker for risk assessment of death, recurrent myocardial infarction and congestive heart failure. It was further analyzed whether these risks could be improved by statins. According to Bonaca, GDF-15 is not a suitable marker for the therapeutic efficacy of statin therapy.
WO 09/047283 discloses a method for deciding which treatment or combination of treatments including statin treatment is applied to the remodeling process in a patient after myocardial infarction, which is based on the detection of three markers: natriuretic peptides, cardiac troponin, and inflammatory markers such as GDF-15. However, the document does not relate to stratification (stratification) of treatment of heart failure patients with statins.
US 2011/0065204 discloses a method for identifying a patient's sensitivity to heart failure treatment, which is based on the quantification of GDF-15 in a sample of a patient suffering from heart failure. Statin therapy is mentioned as a possible treatment of the patients enrolled in the study described in the examples, but not for heart failure. Furthermore, GDF-15 is not disclosed as a marker that can be used to identify a patient as likely to or likely not to respond to statin therapy.
WO 2009/138451 discloses a method for deciding which statin drug to use for a seemingly stable patient suffering from heart failure and experiencing a change in physiological state, the method comprising repeatedly determining the amount of the peptide markers NT-proANP, NT-proBNP, cardiac troponin and GDF-15 in a patient sample over a given time interval.
Sola et al (J Card fail. 2005 Oct;11 (8):607-12) disclose that in heart failure patients treated with statins, a decrease in IL-6 levels indicates that statins exert a positive influence on the inflammatory process. The publication does not disclose that IL-6 can be used for stratification prior to therapeutic application of statins in heart failure patients.
WO 2007/26214 discloses a method of predicting a patient's response to a drug or drug candidate. As one of several drugs, statins are mentioned.
In the context of studies based on the present invention, it was advantageously shown that GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive c-reactive protein) can be used to identify a subset of heart failure patients who respond to statin therapy. In particular, biomarker levels in the blood can predict whether a heart failure patient will benefit from or will be compromised by statin therapy.
Disclosure of Invention
Accordingly, the present invention relates to a method of identifying a patient with heart failure as likely to respond to a treatment comprising a statin, comprising
(a) Measuring the level of at least one biomarker in a sample of the patient selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive c-reactive protein), and
(b) comparing the level of the at least one biomarker to a respective reference level.
In embodiments, the method further comprises step (c): when the level of the biomarker (depending on the respective marker) in the patient sample is above or below a reference level, the patient is identified as more likely or less likely to respond to a treatment comprising statin. Preferred diagnostic algorithms are disclosed elsewhere herein.
In a further embodiment, the method comprises step (d): treatment comprising statins is recommended, initiated or discontinued.
In one embodiment, the level of at least one biological inhibitor is measured by: contacting the sample with a reagent that specifically binds to each label, thereby forming a complex between the reagent and the label, detecting the amount of complex formed, and thereby measuring the level of the label. This is particularly suitable if the biomarker to be measured is a polypeptide (GDF-15, SHBG, PLGF IL-6, transferrin, cardiac troponin, sFlt-1, prealbumin, ferritin, osteopontin, sST2 and hscRP).
If the biomarker is uric acid or urea, the level of said biomarker is preferably measured by contacting the sample with an enzyme of a compound that allows the conversion of said biomarker.
If the biomarker being measured is uric acid, the level of said biomarker is preferably measured by contacting the sample with a compound or enzyme that allows the oxidation of uric acid. The enzyme is preferably uricase (EC 1.7.3.3), which catalyzes the oxidation of uric acid to 5-hydroxyisocyanuric acid. The compound is preferably phosphotungstic acid.
If the biomarker being measured is urea, the level of the biomarker is preferably measured by contacting the sample with a urease (EC 3.5.1.5) that catalyzes the hydrolysis of urea to carbon dioxide and ammonia. In addition, the sample may subsequently be contacted with glutamate dehydrogenase (EC 1.4.1.2). In a second reaction, 2-ketoglutarate reacts with ammonia in the presence of glutamate dehydrogenase (GLDH) and coenzyme NADH to produce L-glutamic acid.
The methods of the invention are preferably ex vivo or in vitro methods. Further, it may comprise steps other than those explicitly mentioned above. For example, further steps may involve sample pre-treatment or evaluation of the results obtained by the method. The method can be carried out manually or with the aid of automation. Preferably, steps (a) and/or (b) may be wholly or partially assisted by automation, e.g. for determination in step (a) by suitable machinery and sensor equipment or for computer-implemented comparison in step (b) and/or evaluation based on said comparison.
In the context of the methods of the invention, a patient is identified as likely to respond to a treatment comprising a statin, i.e., a treatment comprising administration of one statin (or administration of more than one statin). Preferably, the statin is administered orally.
Statins are well known in the art. Statins (also commonly referred to as "HMG-CoA reductase inhibitors") are useful in inhibiting the enzyme: HMG-CoA reductase, a class of drugs that lowers cholesterol levels, plays an important role in the production of cholesterol in the liver. By inhibiting HMG-CoA reductase, statins block the pathway of synthesizing cholesterol in the liver. This is important because most circulating cholesterol is derived from internal manufacturing rather than from diet.
Statins are divided into two groups, i) fermentatively derived and ii) synthetic. In the context of the present invention, statins may be either synthetic or fermentatively derived. Preferred statins are selected from the following: atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin. In a particularly preferred embodiment the statin is atorvastatin or pravastatin.
The phrase "identifying a patient" as used herein preferably refers to using information or data generated relating to the level of at least one marker referred to herein in a patient sample to identify or select patients more likely or less likely to benefit from a treatment comprising statin. In particular, a patient is considered to be responsive to (and therefore more likely to benefit from) a treatment comprising a statin if the treatment reduces the risk of death of the patient and/or reduces the risk of hospitalization of the patient (preferably within a window period of 18 months or 3 years after the sample being tested has been obtained). Preferably, one or more of the above mentioned risks is reduced by at least 5%, more preferably by at least 10% and most preferably by 20%. Moreover, a patient is considered to not respond to (and therefore less likely to benefit from) a treatment comprising a statin if the treatment does not reduce the risk of mortality and/or hospitalization, in particular does not significantly reduce the risk of mortality and/or hospitalization of the patient (preferably within a window period of 18 months or 3 years after the sample tested has been obtained). In this case, unnecessary health care costs can be avoided if the drug is not administered. Further, adverse side effects resulting from treatments involving statins can be avoided.
The terms "death" and "hospitalization" are defined herein.
The information or data used or generated for authentication may be in any form, written, spoken or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, sending, transmitting, providing, propagating, distributing, or a combination thereof. In some embodiments, the communicating, presenting, reporting, storing, sending, transmitting, providing, propagating, distributing, or a combination thereof is performed by a computer device, an analyzer unit, or a combination thereof. In some further embodiments, the communicating, presenting, reporting, storing, sending, transmitting, providing, propagating, distributing, or a combination thereof is performed by a laboratory or medical professional. In some embodiments, the information or data comprises a comparison of the level of the at least one marker to a reference level. In some embodiments, the information or data comprises information indicating that the patient is more or less likely to respond to a therapy comprising a statin.
The terms "less likely" and "more likely" are understood by the skilled person. A patient who is more likely to respond to a treatment comprising a statin has an increased likelihood, in particular a significantly increased likelihood, to said treatment compared to the average likelihood of the patient population, whereas a patient who is less likely to respond to a treatment comprising a statin has a decreased likelihood, in particular a significantly decreased likelihood, to said treatment compared to the average likelihood of the patient population. Preferably, the population of patients shows the same characteristics. In particular, it is envisaged that the patient is included in a population with heart failure. Further, as explained elsewhere herein, the patient may or may not have coronary artery disease (which may depend on the measured biomarker). By increased likelihood is meant that the likelihood is preferably increased by at least 10%, more preferably at least 20% or 30% and most preferably at least 40% compared to the average likelihood in a patient population. By reduced likelihood is meant that the likelihood is preferably reduced by at least 10%, more preferably by at least 20% or 30% and most preferably by at least 40% compared to the average likelihood in the patient population.
As will be appreciated by those skilled in the art, assessing whether a patient is likely to respond to a therapy that includes a statin is generally not intended to be 100% correct for the patient being assessed. However, this term requires that the assessment be correct for a statistically significant portion of patients (e.g., cohorts in a cohort study). Whether a moiety is statistically significant can be determined by one skilled in the art without further effort using various well-known statistical evaluation tools, such as determination of 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, 0.1, 0.05, 0.01, 0.005 or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the patient population can be suitably identified by the method of the invention.
The terms "patient" and "subject" are used interchangeably herein. The term as used herein in the context of the above methods relates to an animal, preferably a mammal, and more preferably a human. A "patient" or "subject" herein is preferably any individual human subject eligible for treatment who is experiencing or has experienced one or more signs, symptoms or other indicators of heart failure. It is intended to include as subjects any subjects involved in clinical research trials, or subjects involved in epidemiological studies, or subjects once used as controls. The patient may have been previously treated with statin, or not. Thus, the patient being tested may have been treated with statin prior to obtaining the sample to be tested, or may not have been treated with statin prior to obtaining the sample.
It is envisaged in the context of the present invention that the patient suffers from Heart Failure (HF), in particular symptomatic heart failure.
The term "heart failure" as used herein relates to impaired systolic and/or diastolic function of the heart, which is accompanied by obvious signs of heart failure as known to those skilled in the art. Preferably, heart failure is also referred to herein as chronic heart failure. Heart failure according to the present invention includes overt heart failure and/or advanced heart failure. In overt heart failure, patients exhibit heart failure symptoms known to those skilled in the art.
HF can be classified to various degrees of severity.
According to the NYHA (New York Heart Association) classification, Heart failure patients may be classified as belonging to NYHA types I, II, III and IV. A patient with heart failure has experienced structural and functional changes in his pericardium, myocardium, coronary circulation, or heart valves. He will not be able to fully restore his health and will need therapeutic treatment. Patients of NYHA type I have no obvious symptoms of cardiovascular disease, but already have objective evidence of impaired function. Patients of NYHA type II have slight limitations of physical activity. Patients of NYHA type III show significant limitations of physical activity. Patients of NYHA type IV cannot perform any physical activity without discomfort. They showed symptoms of cardiac insufficiency at rest.
This functional classification is supplemented by the newer classification of the American College of medicine and the American Heart Association (ACC/AHA classification, see J. Am. Col. Cardiol. 2001;38; 2101-. 4 phases A, B, C and D are defined. Phases a and B are not HF but are believed to help identify patients earlier before progressing to "true" HF. Phase a and B patients are best defined as patients with risk factors for HF development. For example, a patient with coronary artery disease, hypertension, or diabetes who has not demonstrated impaired Left Ventricular (LV) function, hypertrophy, or geometric ventricular deformity will be considered stage a, while a patient who is asymptomatic but demonstrates LV hypertrophy and/or impaired LV function will be designated stage B. Then phase C represents patients with symptoms of HF currently or historically associated with an underlying structural heart disease (most patients with HF), and phase D designates patients with truly refractory HF.
As used herein, the term "heart failure" refers specifically to stages B, C and D indicated by the ACC/AHA classification above. During these phases, the patient shows typical symptoms of heart failure and/or shows structural and/or functional abnormalities of the heart. Thus, a patient with heart failure, according to the ACC/AHA classification, has heart failure classified as stages B, C and D. Also preferably, the patient has heart failure classified as NYHA type II, III or IV. In preferred embodiments, the term "heart failure" refers to stages B and C of the ACC/AHA classification referred to above, or heart failure classified as NYHA type II or type III. Thus, the patient preferably suffers from heart failure classified as stage B or C according to the ACC/AHA classification. Also preferably, the patient has heart failure classified as NYHA type II or III.
In addition to heart failure, the patient may or may not have coronary artery disease, depending on the biomarker being measured.
If the biomarker is transferrin or ferritin, the patient preferably suffers from heart failure and coronary artery disease.
If the biomarker is osteopontin or sST2, the patient preferably suffers from heart failure but preferably does not suffer from coronary artery disease.
If the biomarker is GDF-15, urea, uric acid, cardiac troponin, SHBG, prealbumin, PlGF, sFlt-1, IL-6, or hscRP, the patient may or may not have coronary artery disease. However, where the biomarker is urea, uric acid, sFlt-1, PlGF, or IL-6, preferably the patient also has CAD.
The term "coronary artery disease", abbreviated CAD, also often referred to as Coronary Heart Disease (CHD) or atherosclerotic heart disease, is known to those skilled in the art. Preferably, the term refers to a condition in which blood and oxygen supply to the blood vessels of the heart narrows. Coronary artery disease is often caused by a condition known as atherosclerosis, which occurs when fatty substances and substances known as plaque build up on your artery wall. Which causes their lumens to narrow. In particular, CAD is the result of the accumulation of atheromatous plaques in the wall of the arteries supplying the heart muscle (heart muscle). Preferably, a patient with stable CAD has at least 50% stenosis (and thus at least 50% occlusion) in at least one major coronary artery. How to assess the degree of coronary occlusion is well known in the art, preferably the degree is assessed by coronary angiography. Whereas the symptoms and signs of coronary artery disease are noted in the late stages of the disease, many individuals with coronary artery disease show no signs of disease for decades, as the disease progresses and eventually occurs before the first appearance of symptoms of an acute event (usually a "sudden" heart attack).
If the patient also has coronary artery disease, it is particularly contemplated that the patient has stable coronary artery disease. The term "stable" in this context means that the patient does not suffer from ACS (acute coronary syndrome), in particular when the sample to be tested has been obtained. The term "ACS" is well known in the art and includes STEMI (ST-elevated myocardial infarction); NSTEMI (non-ST-elevated myocardial infarction) and unstable angina. It is further expected that the patients tested do not have a recent history of ACS and therefore should not have an ACS in the near future. In particular, the patient should not suffer from ACS within the month before carrying out the method of the invention (more precisely, within the month before obtaining the sample).
Preferably, the patient in the context of the present invention does not have impaired renal function. Preferably, the patient should not suffer from renal failure, in particular the patient should not suffer from acute, chronic and/or end-stage renal failure. Further, the patient should preferably not suffer from renal hypertension. How to assess whether a patient exhibits impaired renal function is well known in the art. Renal disorders can be diagnosed by any means known and deemed appropriate. In particular, renal function can be assessed by way of Glomerular Filtration Rate (GFR). For example, GFR can be calculated 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 bowden capsule per unit time. Clinically, this is often used to determine renal function. All derived from the calculated delivery estimates of formulas, such as the Cockgroft Gault formula or MDRD formula and not the "true" GFR, by injection of inulin into plasma. Because inulin is not reabsorbed by the kidney after glomerular filtration, its rate of excretion is directly proportional to the filtration rate of water and solutes passing through the glomerular filter. However, in clinical practice, creatinine clearance is used to measure GFR. Creatinine is an endogenous molecule that is synthesized in the body, which is freely filtered by the glomerulus (but is also secreted in small amounts by the tubules). Creatinine clearance (CrCl) is therefore a close approximation of GFR. GFR is typically recorded in milliliters per minute (mL/min). The normal range of GFR is 97 to 137 mL/min for men and 88 to 128 mL/min for women. Thus, it is particularly contemplated that GFR for patients who do not exhibit impaired renal function is within this range. Furthermore, the subject preferably has a blood creatinine level (particularly serum creatinine level) of less than 0.9 mg/dl, more preferably less than 1.1 mg/dl and most preferably less than 1.3 mg/dl.
The term "sample" refers to a sample of bodily fluid, refers to a sample of isolated cells or a sample from a tissue or organ. Samples of body fluids may be obtained by well-known techniques and include samples of blood, plasma, serum, urine, lymph, saliva, ascites, bronchial lavage or any other bodily secretion or derivative thereof. Tissue or organ samples may be obtained from any tissue or organ by, for example, biopsy. Isolated cells may be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. For example, a cell-, tissue-, or organ sample can be obtained from those cells, tissues, or organs that express or produce a biomarker. The sample may be frozen, fresh, fixed (e.g., formalin fixed), centrifuged, and/or embedded (e.g., paraffin embedded), and the like. The cell sample may, of course, be subjected to a variety of well-known post-collection preparation and storage techniques (e.g., nucleic acid and/or protein extraction, immobilization, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of marker in the sample. Likewise, biopsies can also undergo post-collection preparation and storage techniques, such as fixation.
In a preferred embodiment of the invention, the sample is a blood, serum or plasma sample. In particular, it is contemplated to measure the level of a biomarker in a plasma sample.
In the context of the present invention, the level of at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive c-reactive protein). Thus, the level of a single biomarker, or the level of a combination of biomarkers, can be measured.
In a preferred embodiment, the level of at least one biomarker selected from GDF-15, urea, SHBG, PLGF, or IL-6 is measured.
In the following, definitions of biomarkers used in the context of the present invention are given.
GDF-15 initially has been cloned as
The term "cardiac troponin" also encompasses variants of the specific troponins mentioned above, i.e. preferably troponin I, and more preferably troponin T. Such variants have at least the same essential biological and immunological properties as the particular cardiac troponin. In particular, they share the same essential biological and immunological properties if they can be detected by the same specific assay indicated in the present description (for example, by an ELISA assay using polyclonal or monoclonal antibodies that specifically recognize said cardiac troponin). Furthermore, it is to be understood that the variants indicated according to the invention should have an amino acid sequence which differs due to substitution, deletion and/or addition of at least one amino acid, wherein the amino acid sequence of the variant also preferably has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98% or at least about 99% identity with the amino acid sequence of the particular troponin. Variants may be allelic variants or specific homologues, orthologues or orthologues of any other species. Furthermore, the variants referred to herein include fragments of the particular cardiac troponin or of the type of variants mentioned above, as long as these fragments have the necessary immunological and biological properties referred to above. Preferably, the cardiac troponin variant has immunological properties (i.e. epitope composition) comparable to those of human troponin T or troponin I. Thus, the variant should be identifiable by the above-mentioned method or by a ligand for determining the concentration of cardiac troponin. Thus, the variant should be identifiable by the above-mentioned method or by a ligand for determining the concentration of cardiac troponin. Such fragments may be, for example, degradation products of troponin. Further included are variants that differ due to post-translational modifications, such as phosphorylation or myristylation. Preferably the biological property of troponin I and variants thereof is the ability to inhibit actomyosin ATPase or to inhibit angiogenesis in vivo and in vitro, which can be detected, for example, based on the assay described by Moses et al 1999 PNAS USA 96 (6): 2645-. Preferably the biological property of troponin T and variants thereof is the ability to form complexes with troponin C and I, to bind calcium ions or to bind tropomyosin, preferably present as a complex of troponin C, I and T or a complex formed from troponin C, troponin T and variants of troponin T. It is known that low concentrations of circulating cardiac troponin can be detected in subjects under a variety of conditions, but further studies are required to understand their respective effects and ratios (Masson et al, Curr Heart Fail Rep (2010) 7: 15-21).
Osteopontin (OPN), also known as bone sialoprotein I (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP 1), 2ar, and rickettsial resistance (Ric), are polypeptides that are highly negatively charged, extracellular matrix proteins that lack extensive secondary structure. It consists of about 300 amino acids (297 in mouse, 314 in human) and is expressed as a 33-kDa nascent protein; there are also functionally important cleavage sites. OPN can undergo post-translational modifications, which increase its apparent molecular weight to about 44 kDa. The sequence of osteopontin is well known in the art (human osteopontin: UniProt P10451, GenBank NP-000573.1), which is found in normal plasma, urine, milk and bile (US 6,414,219; US 5,695,761; Denhardt, D.T. and Guo, X., FASEB J. 7 (1993) 1475-1482; Oldberg, A., et al, PNAS 83 (1986) 8819-8823; Oldberg, A., et al, J. biol. chem. 263 1988) 19433-19436; Giachelli, CM., et al, Trends Cardiovasc. Med. 5(1995) 88-95). Human OPN proteins and cDNAs have been isolated and sequenced (Kiefer M. C, et al, Nucl. acids sRs. 17 (1989) 3306). OPN plays a role in cell adhesion, chemotaxis, macrophage directed interleukin-10. OPN is known to interact with several integrin receptors. Increased OPN expression has been reported in several human cancers, and its cognate receptors have been identified (av-b 3, av-b5, and av-bl integrins and CD 44). In vitro studies by Irby, r.b., et al, clin.exp. Metastasis 21 (2004) 515-523, indicated that both endogenous OPN expression (by stable transfection) and exogenous OPN (added to the culture medium) increased the mobility and invasiveness of human colon cancer cells in vitro.
The term "soluble Flt-1" or "sFlt-1" as used herein refers to a polypeptide which is a soluble form of the
The term "sFlt-1" as used herein also encompasses variants of the specific sFlt-1 polypeptides mentioned above. Such variants have at least the same essential biological and immunological properties as the particular sFlt-1 polypeptide. In particular, they share the same essential biological and immunological properties if they can be detected by the same specific assay indicated in the present specification (e.g., by an ELISA assay using a polyclonal or monoclonal antibody specifically recognizing said sFlt-1 polypeptide). For a more detailed explanation of the term "variant" please see above.
The term "PlGF" (placental growth factor) as used herein refers to a placenta-derived growth factor that is a polypeptide of 149 amino acids in length and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human Vascular Endothelial Growth Factor (VEGF). Such as VEGF, PlGF has angiogenic activity in vitro and in vivo. For example, biochemical and functional characterization of PlGF derived from transfected COS-1 cells shows that it is a glycosylated dimeric secretory protein capable of stimulating endothelial cell growth in vitro (Maqlione 1993, Oncogene 8(4): 925-31). Preferably, PlGF refers to human PlGF, more preferably human PlGF having the amino acid sequence set forth in Genbank accession number P49763, GI: 17380553.
ST2, frequently also referred to as "
Interleukin-6 (abbreviated IL-6) is an interleukin secreted by T cells and macrophages to stimulate an immune response, for example during infection and after trauma, in particular burns or other tissue damage leading to inflammation. It acts as a pro-inflammatory and anti-inflammatory cytokine. In humans, it is encoded by the IL6 gene. The sequence of human IL-6 can be evaluated by GenBank (see NM _000600.3 for polynucleotide sequences, and NP _000591.1 for amino acid sequences). IL-6 signals through a cell surface class I cytokine receptor complex consisting of a ligand-bound IL-6R α chain (CD126) and the signal transduction component gp130 (also known as CD 130). CD130 is a common signal transduction protein for several cytokines including Leukemia Inhibitory Factor (LIF), ciliary neurotrophic factor, oncostatin M, IL-11, and cardiotrophin-1, and it is expressed almost ubiquitously in most tissues. In contrast, expression of CD126 is restricted to certain tissues. As IL-6 interacts with its receptor, it triggers gp130 and IL-6R proteins to form a complex, thereby activating the receptor. These complexes, along with the intracellular region of gp130, initiate a signaling cascade through certain transcription factors, tyrosine kinases (JAKs), and signal transduction proteins and transcriptional activators.
CRP, also referred to herein as C-reactive protein, is an acute phase protein that was discovered more than 75 years ago as a blood protein that binds the C-polysaccharide of pneumococci (pneumococci). CRP is known as a marker of reactive inflammation and is produced by remote organs (i.e., the liver) in response to or in response to chemokines or interleukins originating from major sites of pathology. CRP consists of five signal subunits, which are non-covalently linked and assembled as a circular pentamer with a molecular weight of approximately 110-140 kDa. Preferably, CRP as used herein relates to human CRP. The sequences of human CRP are well known and are disclosed, for example, in Woo et al (J.biol. chem. 1985.260 (24), 13384-13388). CRP levels are usually lower in normal individuals, but may be increased by 100-fold or more due to inflammation, infection or injury (Yeh (2004) circulation. 2004; 109: II-11-II-14). CRP is known to be an independent factor for cardiovascular risk prediction. In particular, CRP has been shown to be suitable as predictor for myocardial infarction, stroke, peripheral arterial disease and sudden cardiac death. In addition, elevated CRP levels can also predict recurrent ischemia and death in patients with Acute Coronary Syndrome (ACS) and patients undergoing coronary intervention. CRP determination is recommended by expert groups (e.g., by the American Heart Association) in patients at risk for coronary Heart disease (see also Pearson et al (2003) Markers of Inflammation and cardiovascular disease Circulation, 107: 499-. The term CRP also relates to variants thereof.
Preferably, the amount of CRP in the patient sample is determined by using a CRP assay with high sensitivity. Determination of CRP by such an assay is also often referred to as high-sensitive CRP (hscrp). hsCRP assays, for example, are used to predict risk of heart disease. Suitable hsCRP assays are known in the art. A particularly preferred hscRP assay in the context of the present invention is the Roche/Hitachi CRP (Latex) HS test with a detection limit of 0.1 mg/l.
Ferritin is a ferric storage protein. Ferritin is a macromolecule with a molecular weight of at least 440 kDa, which depends on the iron content, and is composed of a protein shell of 24 subunits (apoferritin) and a protein shell containing an average of about 2500 Fe3+Iron core composition of ions (in liver and spleen ferritin). In vertebrates, these subunits are both light (L) and heavy (H) forms, having an apparent molecular weight of 19 kDA or 21kDA, respectively. Ferritin tends to form oligomers. At least 20 of the iso-transferrins can be distinguished using isoelectric focusing. These microscopic inhomogeneities are due to differences in acidic H and weakly basic L subunit contents. Basic transferrin is responsible for long-term iron storage function and is found mainly in the liver, spleen and bone marrow.
Prealbumin is a tryptophan-rich protein which is synthesized in hepatocytes and has a molar mass of 55 kDa. Due to its greater rate of dispersion towards the anode, an electrophoretic band appeared < 2.5% relative amount before albumin at pH 8.6. Its function is to bind and transport low molecular weight retinol binding proteins (less than 21kDa molar mass), preventing their glomerular filtration. 30-50% of the circulating prealbumin forms a complex with retinol binding protein. In addition, it binds and transports thyroxine (T4). Frequently, prealbumin is also known as transthyretin.
Sex Hormone Binding Globulin (SHBG) is the blood transport protein for testosterone and estradiol. It is a large glycoprotein with a molecular weight of about 95 kD and exists as a homodimer composed of two identical subunits. Each subunit contains two disulfide bonds. SHBG is mostly produced by the liver and released into the bloodstream. Other sites for SHBG production include brain, uterus, testis, and placenta. The sequence of SHBG is well known in the art, see, e.g., GenBank accession No. NP-001031.2 GI: 7382460).
Transferrin is a glycoprotein having a molecular weight of about 80 kDa. It comprises polypeptide chains with two N-glycoside-linked oligosaccharide chains and exists in multiple isoforms. The rate of synthesis in the liver can vary depending on the iron demand and iron inventory in the body. Transferrin is an iron transport protein in serum. In the case of iron deficiency, the degree of transferrin saturation appears to be a very sensitive indicator of functional iron deficiency. A variety of methods are available for the determination of transferrin, including radioimmunodiffusion, nephelometry and turbidimetry.
In the context of the method of the invention, it is particularly envisaged to determine the amount of human peptide or polypeptide.
Uric acid is the end product of purine metabolism in the host organism. The IUPAC name is 7, 9-dihydro-3H-purine-2, 6, 8-trione. The compounds are often referred to as uric acid (urate), uric acid (lithoic acid), 2,6, 8-trioxypurine, 2,6, 8-trihydroxypurine, 2,6, 8-trioxypurine, 1H-purine-2, 6, 8-triol (formula C)5H4N4O3PubChem CID 1175, CAS number 69-93-2).
Uric acid measurements are used to diagnose and treat a variety of renal and metabolic disorders, including renal failure, gout, leukemia, psoriasis, hunger or other wasting conditions, and patients receiving cytotoxic drugs. The oxidation of uric acid provides the basis for two methods for quantitatively determining this purine metabolism. One method is to reduce phosphotungstic acid to tungsten blue in an alkaline solution, which is measured by photometric measurements. The second method is described by Praetorius and Poulson, which uses the enzyme uricase to oxidize uric acid; the method eliminates the interference inherent to chemical oxidation (Praedius E, Poulsen H. Enzymatic Determination of Uricacid with purified orientations. Scandinav J Clin Lab interrogation 1953: 273-. Uricase may be used in methods involving UV measurement of uric acid consumption or in combination with other enzymes to provide a colorimetric assay. Another method is a colorimetric method developed by Town et al (Town MH, Gehm S, Hammer B, Ziegenhorn J. J ClinChem Clin Biochem 1985;23: 591). The sample is initially incubated with a reagent mixture containing ascorbate oxidase and a transparent system. It is important in this test system that any ascorbic acid present in the sample is eliminated in the initial reaction; this excludes any ascorbic acid interfering with the subsequent POD indicator reaction. After addition of the initial reagents, the oxidation of uric acid by uricase is started.
In the context of the present invention, uric acid may be determined by any method deemed suitable. Preferably, the biomarkers are determined by the methods mentioned above. More preferably, uric acid is determined by applying a slightly modified colorimetric method as described above. In this reaction, a peroxide is reacted in the presence of Peroxidase (POD), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS) and 4-aminopyrine to form a quinone diimine dye. The intensity of the red color formed is directly proportional to the uric acid concentration and is determined photometrically.
Urea is the major end product of protein nitrogen metabolism. Which has the chemical formula CO (NH)2)2And is synthesized from ammonia produced by deamination of amino acids through the urea cycle in the liver. Urea is mostly excreted through the kidneys, but small amounts are also excreted through sweat and degraded in the gut by bacterial action. The determination of blood urea nitrogen is the most widely used renal function screening test.
The term "measuring" a level of a biomarker as used herein refers to quantification of the biomarker, e.g., determining the level of the biomarker in a sample using a suitable detection method described elsewhere herein. The terms "measuring", "detecting" and "determining" are used interchangeably herein.
The biomarkers referred to herein may be detected using methods generally known in the art. Detection methods generally encompass methods of quantifying the level of a biomarker in a sample (quantification methods). The following methods, which are suitable for the qualitative and/or quantitative detection of biomarkers, are generally known to the person skilled in the art. Samples can be conveniently assayed, for example using western blots and immunoassays for proteins, such as ELISA, RIA, fluorescence based immunoassays, which are commercially available. Detecting biomarkersFurther suitable methods of measuring a particular physical or chemical property of a peptide or polypeptide include measuring its exact molecular weight or NMR spectrum. The methods include, for example, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass spectrometers, NMR-analyzers, or chromatographic devices. Further, methods include microplate ELISA based methods, fully automated or robotic immunoassays (e.g., as available in Elecsys)TMObtained from an analyzer), CBA (enzyme cobalt binding assay, e.g.as can be found in Roche-HitachiTMObtained on an analyzer) and latex agglutination assays (e.g., as may be found in Roche-HitachiTMObtained by an analyzer).
For biomarker protein detection as referred to herein, a wide range of immunoassay techniques using such assay formats are available, see, e.g., U.S. patent nos. 4,016,043, 4,424,279, and 4,018,653. These include single and double-site or "sandwich" assays of the non-competitive type, as well as traditional competitive binding assays. These assays may include direct binding of labeled antibodies to the target biomarkers.
Of these, the sandwich assay is the most useful and commonly used immunoassay.
Methods for measuring electrochemiluminescence phenomena are well known. Such methods use the ability of specific metal complexes to achieve excited states from which they decay from a ground state to emit electrochemiluminescence by oxidation. For a review see Richter, M.M., chem. Rev. 104 (2004) 3003-3036.
Biomarkers can also be detected by commonly known methods including magnetic resonance spectroscopy (NMR spectroscopy), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), high and ultra-HPLC, such as reverse phase HPLC, e.g., ion-pairing HPLC with dual UV wavelength detection, capillary electrophoresis with laser induced fluorescence detection, anion exchange chromatography and fluorescence detection, thin layer chromatography.
Preferably, measuring the level of the peptide or polypeptide comprises the steps of: (a) contacting a cell capable of eliciting a cellular response, the intensity of which is indicative of the level of the peptide or polypeptide, with said peptide or polypeptide for a sufficient time, (b) measuring the cellular response. For measuring cellular responses, the sample or treated sample is preferably added to a cell culture and either internal or external cellular responses are measured. The cellular response may include expression of a measurable reporter gene or secretion of a substance, such as a peptide, polypeptide, or small molecule. The expression or substance should generate an intensity signal which correlates with the level of the peptide or polypeptide.
Also preferably, measuring the level of the peptide or polypeptide comprises the step of measuring a specific intensity signal obtainable from the peptide or polypeptide in the sample. As described above, such a signal may be the signal intensity specific for the peptide or polypeptide observed in the m/z variation, which is observed in a mass spectrum or NMR spectrum specific for the peptide or polypeptide.
Measuring the level of the peptide or polypeptide preferably comprises the steps of (a) contacting the peptide with a specific binding agent, (b) (optionally) removing unbound binding agent, (c) measuring the level of bound binding agent, i.e. the complex of binding agent formed in step (a). According to a preferred embodiment, said steps of contacting, removing and measuring may be performed by an analyzer unit of the system described herein. According to some embodiments, the steps may be performed by a single analyzer unit of the system or by more than one analyzer unit in operable communication with each other. For example, according to certain embodiments, the systems disclosed herein may include a first analyzer unit for performing the steps of contacting and removing, and a second analyzer unit operatively connected with the first analyzer unit via a transfer unit (e.g., robotic arm), the second analyzer unit performing the steps of measuring.
The bound binding agent, i.e. the binding agent or binding agent/peptide complex, will generate an intensity signal. Binding according to the present invention includes both covalent and non-covalent binding. A binding agent according to the invention may be any compound, such as a peptide, polypeptide, nucleic acid, or small molecule, which binds to a peptide or polypeptide described herein. Preferred binding agents include antibodies, nucleic acids, peptides or polypeptides, such as receptors or binding partners for peptides or polypeptides and fragments thereof comprising the peptide binding domain, and aptamers, e.g., nucleic acid or peptide aptamers. Methods of making such binders are well known in the art. For example, the identification and production of suitable antibodies or aptamers is also provided by commercial suppliers. The person skilled in the art is familiar with methods for developing derivatives of such binding agents with higher affinity or specificity. For example, random mutations can be introduced into a nucleic acid, peptide, or polypeptide. These derivatives can then be tested for binding according to screening procedures known in the art, such as phage display. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof such as Fv, Fab and f (ab)2 fragments, which are capable of binding to an antigen or hapten. The invention also includes single chain antibodies and humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody that exhibits the desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence will typically include at least the antigen binding amino acid residues of the donor, but may also include other structurally and/or functionally relevant amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. Preferably, the binding agent or agents specifically bind to the peptide or polypeptide. Specific binding according to the present invention means that the ligand or agent should not substantially bind to (be "cross-reactive with") another peptide, polypeptide or substance present in the sample being analyzed. Preferably, the specific binding peptide or polypeptide should bind with at least 3-fold higher affinity than any other related peptide or polypeptide, more preferably at least 10-fold higher affinity and even more preferably at least 50-fold higher affinity. Non-specific binding may be tolerable if it can still be clearly distinguished and measured, for example, by its size on a western blot, or by its relatively higher abundance in the sample. Binding of the binding agent can be measured by any method known in the art. Preferably, the method is semi-quantitative or quantitative. Further suitable techniques for determining the polypeptide or peptide are described below.
Binding of the binding agent can be measured directly by, for example, NMR or surface plasmon resonance. The measurement of binding of the binding agent is, according to a preferred embodiment, performed by an analyzer unit of the system disclosed herein. Thereafter, the measured level of binding may be calculated by the computer device of the system disclosed herein. If the binding agent is also provided as a substance of the enzymatic activity of the peptide or polypeptide of interest, the enzymatic reaction product can be measured (e.g., the level of protease is measured by measuring the level of cleaved substrate, e.g., on a western blot). Alternatively, the binding agent may itself exhibit enzymatic properties, and the "binding agent/peptide or polypeptide" complex or binding agent bound by a peptide or polypeptide, respectively, may be contacted with a suitable substrate, allowing detection by intensity signal generation. For measuring the enzyme reaction product, it is preferred that the level 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 period of time. A sufficiently long period of time refers to the time required for a detectable, preferably measurable, level of the product to be produced. In addition to measuring the level of product, the time required for a given (e.g., detectable) level of product to occur may be measured. Third, the binding agent may be coupled covalently or non-covalently to a label that allows detection and measurement of the binding agent. Labeling may be accomplished by direct or indirect methods. Direct labeling involves direct (covalent or non-covalent) coupling of the label to a binding agent. Indirect labeling involves the binding (covalently or non-covalently) of a second binding agent to a first binding agent. The second binding agent should specifically bind to the first binding agent. The second binding agent may be coupled to a suitable label and/or be the target (receptor) of a third binding agent that binds the second binding agent. The use of second, third or higher order binders is often used to increase the signal. Suitable secondary and higher binding agents may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The binding agent or substrate may also be "tagged" using one or more tags known in the art. Such tags may then be targets for higher-order binders. 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 a peptide or polypeptide, the tag is preferably at the N-terminus and/or C-terminus. A typical label is any label that can be detected by a suitable detection method. Suitable labels include gold particles, latex beads, 9, 10-dihydroacridinium ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g., magnetic beads" which include 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 di-amino-benzidine (DAB), 3'-5,5' -tetramethylbenzidine, NBT-BCIP (4-nitrotetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as ready-to-use stocks from Roche Diagnostics), CDP-Star ™ (Amersham Bio-sciences), ECF ™ (Amersham Biosciences). Suitable enzyme-substrate combinations can result in chromogenic reaction products, fluorescence or chemiluminescence, which can be measured according to methods known in the art (e.g., using a light-sensitive film or a suitable photographic system). For measuring the enzymatic reaction, the criteria given above apply analogously. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, texas red, fluorescein, and Alexa dyes (e.g., Alexa 568). Further fluorescent labels are available, for example, from Molecular Probes (Oregon). The use of quantum dots as fluorescent labels is also contemplated. The radiolabel may be detected by any known and suitable method, such as a light-sensitive film or a phosphorescent imager.
The level of peptide or polypeptide can also preferably be determined as follows: (a) contacting a solid support comprising a binding agent for a peptide or polypeptide as specified above with a sample comprising the peptide or polypeptide and (b) measuring the level of peptide or polypeptide bound to the support. A binding agent, preferably selected from the following: nucleic acids, peptides, polypeptides, antibodies and aptamers, preferably in immobilized form, on a solid support. Materials for preparing solid supports are well known in the art and include, inter alia: 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 trays, plastic tubes, and the like. The binding agent or reagents may be bound to a variety 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 of immobilizing/immobilizing the binding agent are well known and include, but are not limited to, ionic interactions, hydrophobic interactions, covalent interactions, and the like. Also contemplated according to the invention is the use of "suspended arrays" as arrays (Nolan 2002, Trends Biotechnol. 20(1): 9-12). In such a suspension array, a carrier such as a microbead or microsphere is suspended. The array consists of different microbeads or microspheres, which may be labeled, carrying different binders. Methods for producing such arrays, e.g. based on solid phase chemical or photolabile protecting groups, are generally known (US 5,744,305).
In an embodiment of the method of the invention, the level of the biomarker referred to herein is measured by using the assay described in the examples section.
In another embodiment of the method of the invention, the measurement in step a) may be carried out by an analyzer unit, in particular by an analyzer as defined elsewhere herein.
The term "binding agent" refers to a molecule comprising a binding moiety that specifically binds to the respective biomarker.
The term "specifically binds" or "specifically binds" refers to a binding reaction in which the binding pair molecules exhibit mutual binding under conditions in which they do not significantly bind to other molecules. The term "specifically binds" or "specifically binds" when referring to a protein or peptide as a biomarker refers to a binding reaction in which the binding agent binds to at least 10-7The affinity of M binds to the corresponding biomarker. The term "specifically binds" or "specifically binds" preferably means at least 10 to its target molecule-8M or even more preferably at least 10-9Affinity of M. The term "specific" or "specifically" is used to indicate that present in a sampleOther molecules do not significantly bind to a binding agent specific for the target molecule. Preferably, the level of binding of molecules that are not target molecules results in a binding affinity that is only 10% or less, more preferably only 5% or less, of the affinity for the target molecules.
Examples of "binding agents" are nucleic acid probes, nucleic acid primers, DNA molecules, RNA molecules, aptamers, antibodies, antibody fragments, peptides, Peptide Nucleic Acids (PNA) or compounds. Preferred binding agents are antibodies that specifically bind to the biomarker being measured. The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Preferably, the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody.
In one aspect, another binding agent that may be used may be an aptamer that specifically binds to at least one biomarker in a sample. The term "specifically binds" or "specifically binds" when referring to a nucleic acid aptamer as a binding agent refers to a binding reaction in which the nucleic acid aptamer binds to the corresponding target molecule with an affinity in the low nM to pM range.
In yet another aspect, the sample is removed from complexes formed between the binding agent and the at least one label prior to measuring the amount of complex formed. Thus, in one aspect, the binding agent may be immobilized on a solid support. In yet another aspect, the sample can be removed from the complex formed on the solid support by application of a wash solution. The complex formed should be proportional to the amount of the at least one label present in the sample. It will be appreciated that the specificity and/or sensitivity of the binding agent to be applied defines the extent to which the proportion of the at least one label capable of being specifically bound is contained in the sample. Further details of how the determination is carried out may also be found elsewhere herein. The amount of complex formed should be converted to an amount of at least one label that reflects the amount actually present in the sample. In one aspect, such an amount may be substantially the amount present in the sample or may be, in another aspect, the amount of some portion thereof (due to the relationship between the amount present in the formed complex and the original sample).
The term "level" as used herein encompasses the absolute amount of a biomarker as indicated herein, the relative amount or concentration of the biomarker, and any value or parameter associated therewith or derivable therefrom. Such values or parameters include intensity signal values of all specific physical or chemical properties obtained from the peptide by direct measurement, for example intensity values in a mass spectrum or NMR spectrum. Furthermore, all values or parameters obtained from direct measurements as described elsewhere in the specification are covered, e.g. the amount of response determined from a biological readout system in response to the peptide or signal intensity obtained from a specific binding partner. It will be appreciated that values relating to the quantities or parameters mentioned above may also be obtained by standard mathematical operations.
The term "comparing" as used herein refers to comparing the level of a biomarker in a sample of an individual or patient to a reference level of a biomarker described elsewhere in this specification. It is to be understood that comparison as used herein generally refers to comparison of corresponding parameters or values, e.g., an absolute value is compared to an absolute reference level, while a concentration is compared to a reference concentration or an intensity signal obtained from a sample biomarker is compared to the same type of intensity signal obtained from a reference sample. The comparison may be performed manually or with assistance from a computer. Thus, the comparison may be implemented by a computer device (e.g., of the system disclosed herein). The value of the measured or detected level of the biomarker in the individual or patient sample and the reference level may be compared, for example, to each and the comparison may be automatically performed by a computer program executing an algorithm of comparison. A computer program implementing the evaluation will provide the desired assessment in a suitable output form. For a computer-aided comparison, the value of the determined quantity may be compared with a value corresponding to a suitable reference, which reference is stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output form. For a computer-aided comparison, the value of the determined quantity may be compared with a value corresponding to a suitable reference, which reference is stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output form.
The term "reference level" as used herein preferably refers to a predetermined value. In this context "level" encompasses absolute amounts, relative amounts or concentrations, as well as any value or parameter associated therewith or derivable therefrom. As will be appreciated by those skilled in the art, the reference levels are predetermined and are set to comply with conventional requirements, for example in specificity and/or sensitivity. These requirements may vary, for example, from one administrative department to another. It may be exemplified that the assay sensitivity or specificity, respectively, has to be set at certain limits, e.g. 80%, 90%, 95% or 98%, respectively. These requirements may also be defined as positive or negative predictive values. Nevertheless, based on the teachings given herein, it will always be possible for the skilled person to reach the reference values to meet those requirements. In one embodiment, the reference level is determined in a reference sample of a healthy individual. In one embodiment the reference level has been predetermined in a reference sample of the individual disease to which the patient belongs. In certain embodiments, the reference level may be set, for example, to any percentage between 25% and 75% of the overall distribution of values in the individual disease under study. In further embodiments, the reference level may for example be set as the median, the tertile, the quartile determined from the overall distribution of the median of the reference samples for the individual diseases studied. In one embodiment, the reference level may be set as the median determined from the overall distribution of values for the individual diseases studied. The reference level may vary depending on a number of physiological parameters, such as age, sex or subpopulation, and the method used to determine the biomarkers referred to herein. In one embodiment, the reference sample is derived from substantially the same type of cell, tissue, organ or bodily fluid source, such as a sample of an individual or patient undergoing the methods of the invention, e.g., if blood is used as a sample to determine biomarker levels in an individual according to the invention, the reference level is also determined in blood or a portion thereof.
The term "reference level" as used herein preferably refers to a level that allows assigning a patient to a group of patients that are more likely to respond to a treatment comprising a statin, or to a group of patients that are less likely to respond to a treatment comprising a statin. Such a reference level may be a threshold level separating each of these groups. Thus, a reference level of a biomarker as referred to herein should be a level that allows assigning a patient to a group of patients that are more likely to respond to a treatment comprising statin, or to a group of patients that are less likely to respond to a treatment comprising statin. Suitable threshold levels for separating the two groups may be calculated without further trouble by statistical tests as referred to elsewhere herein based on the levels of the markers referred to herein from patients (or from groups of such patients) that are more likely to respond to a treatment comprising statin, or patients (or from groups of such patients) that are less likely to respond to a treatment comprising statin. Preferred reference levels that may be derived from a patient or group of patients as mentioned above are indicated elsewhere herein.
The reference level may be used to define and establish the threshold level. The threshold level preferably allows identification of a patient as more or less likely to respond to a treatment comprising a statin. Thus, the threshold level should preferably allow identification of the patient as more or less likely to respond to a treatment comprising a statin. In one embodiment, the reference level is a calculated reference level. Identification may be provided by the computing device of the system disclosed herein based on said comparison of the calculated "level" to a reference or threshold. For example, the computing device of the system may provide an indicator in the form of a word, symbol, or value that indicates a stroke-in or a stroke-out. The reference level suitable for an individual patient may vary depending on a number of physiological parameters, such as age, sex or subpopulation, and the method used to determine the peptide or polypeptide referred to herein. Suitable reference levels may be determined from the reference sample to be analysed together with the test sample (i.e. simultaneously or sequentially).
Preferably, the reference level is a calculated reference level. Preferably, the calculated reference level should allow to distinguish between patients more likely to respond to a treatment comprising statin and patients less likely to respond to a treatment comprising statin, in principle, the reference level may be calculated for a group of patients as explained above based on the average (average or mean values) of a given biomarker by applying standard statistical methods. In particular, the accuracy of the test, such as a method for diagnosing an event (or not), is best described by its Receiver Operating Characteristics (ROC) (see in particular Zweig1993, Clin. chem. 39: 561-. ROC plots are plots of all sensitivity/specificity pairings resulting from continuously varying decision thresholds for the overall range of data observed. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign a patient to a certain assessment, prognosis or diagnosis. The ROC plot indicates the overlap between the two distributions by plotting the sensitivity against the full range of thresholds applicable for discrimination versus 1-specificity. Sensitivity, or true positive score, is defined on the y-axis as the ratio of the number of true positive test results to the product of the number of true positive test results and the number of false negative test results (product). This also refers to positivity in the presence of a disease or condition. Which are calculated separately from the affected subgroups. The fraction of false positives, or 1-specificity, is defined on the x-axis as the ratio of the number of false positive test results to the product of the number of true negative results and the number of false positive results. It is an indicator of specificity and is calculated entirely from the unaffected subgroup. Because true and false positive scores are calculated entirely separately using test results from two different subgroups, the ROC plot is independent of the prevalence of events in the cohort. Each point in the ROC graph represents a sensitivity/-specificity pair corresponding to a particular decision threshold. The detection with perfect discrimination (no overlap in the two assignments) has a ROC plot through the upper left corner with a true positive score of 1.0, or 100% (perfect sensitivity), and a false positive score of 0 (perfect specificity). The theoretical plot for the test without discrimination (the distribution of results for both groups is the same) is a 45 ° diagonal from the bottom left to the top right. Most of the figures fall between these two extremes. If the ROC graph falls exactly on the 45 ° diagonal, it can be easily remedied by reversing the "positive" criterion from "greater than" to "less than" or vice versa. Qualitatively, the closer the graph is to the upper left corner, the higher the overall accuracy of the test. Depending on the desired confidence interval, the threshold may be derived from a ROC curve, which allows diagnosis or prediction of a given event using an appropriate balance of sensitivity and specificity, respectively. Thus, the reference for the above-mentioned method of the invention may preferably be a threshold or cut-off level and may preferably be generated by establishing an ROC for the population and deriving a threshold level therefrom as described above. ROC plots allow derivation of appropriate thresholds depending on the desired sensitivity and specificity of the diagnostic method.
The following applies as a diagnostic algorithm:
osteopontin as biomarker
If osteopontin is used as a biomarker, the tested heart failure patients preferably do not suffer from coronary artery disease. Preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker above the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
sST2 as biomarker
If sST2 is used as biomarker, the tested heart failure patients preferably do not suffer from coronary artery disease. Preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
GDF-15 as biomarker
If GDF-15 is used as a biomarker, the heart failure patient being tested may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
Urea as biomarker
If urea is used as a biomarker, the tested heart failure patient may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
Uric acid as biomarker
If uric acid is used as a biomarker, the patient being tested may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
Transferrin as a biomarker
If transferrin is used as a biomarker, the patient being tested preferably also suffers from coronary artery disease. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
Cardiac troponin as biomarker
If cardiac troponins, in particular troponin T, are used as biomarkers, the patient to be tested may or may not additionally suffer from coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
SHBG as biomarker
If SHBG is used as the biomarker, the patient being tested may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
sFlt-1 as biomarker
If sFlt-1 is used as a biomarker, the patient being tested may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
Prealbumin as a biomarker
If prealbumin is used as the biomarker, the patient being tested may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
PlGF as biomarkers
If PlGF is used as a biomarker, the patient being tested may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
IL-6 as biomarker
If IL-6 is used as a biomarker, the patient being tested may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
Ferritin as a biomarker
If ferritin is used as a biomarker, the patient being tested preferably also suffers from coronary artery disease. Preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
HsCRP as biomarker
If hsCRP is used as the biomarker, the patient being tested may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
As described above, the reference level may be determined without further trouble. The preferred reference level may be the median level of the group of patients with heart failure.
Preferred diagnostic algorithms for each marker are also disclosed in the section "preferred embodiments", see, e.g., embodiment 8.
Preferred reference levels for GDF-15 are preferably in the range between 2500 pg/mL and 4500 pg/mL, more preferably in the range between 3000 pg/mL and 4000 pg/mL. Most preferably, the reference level is 3560 pg/mL.
The preferred reference level for urea is preferably in the range between 8 mmol/L and 11 mmol/L, more preferably in the range between 9 mmol/L and 10 mmol/L. Most preferably, the reference level is 9.4 mmol/L.
Preferred reference levels for SHBG are preferably in the range between 25 nmol/L and 36 mmol/L, more preferably in the range between 30 nmol/L and 32 mmol/L. Most preferably, the reference level is 30.8 nmol/L.
Preferred reference levels for uric acid are preferably in the range between 6.3 mg/dL and 8.3 mg/dL, more preferably in the range between 6.9 mg/dL and 7.7 mg/dL. Most preferably, the reference level is 7.3 mg/dL.
The preferred reference level for PLGF is preferably in the range between 16 and 26 pg/mL, more preferably between 20 and 23 pg/mL. Most preferably, the reference level is 21.3 pg/mL.
The preferred reference level for IL-6 is preferably in the range between 5.7 pg/mL and 7.1 pg/mL, more preferably in the range between 6.1 pg/mL and 6.7 pg/mL. Most preferably, the reference level is 6.4 pg/mL.
Preferred reference levels for transferrin are preferably in the range between 3.7 g/L and 4.5 g/L, more preferably in the range between 3.9 g/L and 4.3 g/L. Most preferably, the reference level is 4.1 g/L.
Preferred reference levels for troponin are preferably in the range between 20 pg/mL and 31 pg/mL, more preferably between 25 pg/mL and 28 pg/mL. Most preferably, the reference level is 26.5 pg/mL.
Preferred reference levels for sFlt-1 are preferably in the range between 85 pg/mL and 115 pg/mL, more preferably in the range between 93pg/mL and 107 pg/mL. Most preferably, the reference level is 99.6 pg/mL.
Preferred reference levels for prealbumin are preferably in the range between 0.18 g/L and 0.22 g/L, more preferably in the range between 0.19 g/L and 0.21 g/L. Most preferably, the reference level is 0.2 g/L.
The preferred reference level for ferritin is preferably in the range between 140 and 180 mug/L, more preferably in the range between 150 and 170 mug/L. Most preferably, the reference level is 160 μ g/L.
Preferred reference levels for osteopontin are preferably in the range between 85 ng/mL and 115 ng/mL, more preferably in the range between 93ng/mL and 107 ng/mL. Most preferably, the reference level is 100 ng/mL.
Preferred reference levels for sST2 are preferably in the range between 30 ng/mL and 38 ng/mL, more preferably in the range between 32 ng/mL and 36 ng/mL. Most preferably, the reference level is 34 ng/mL.
Preferred reference levels for hsCRP are preferably in the range between 4mg/mL and 6 mg/mL, more preferably in the range between 4.8 mg/mL and 5.4 mg/mL. Most preferably, the reference level is 5.1 mg/mL.
The reference levels given above preferably apply to all methods and uses of the invention.
In certain embodiments, the term "above a reference level" refers to a level of a biomarker in a sample of an individual or patient that is above the reference level or increases by 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or more in total as determined by the methods described herein compared to the reference level. In certain embodiments, the term increase refers to an increase in the level of a biomarker in a sample of an individual or patient, wherein the increase is at least about 1.5-, 1.75-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100-fold greater compared to a reference level (e.g., predetermined from a reference sample).
In certain embodiments, the term "reduce" or "below" herein refers to a level of a biomarker in a sample of an individual or patient that is less than a reference level or is reduced by 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more in total, as determined by the methods described herein compared to a reference level. In certain embodiments, the term decrease in the level of a biomarker in a sample of an individual or patient, wherein the level of decrease is at most about 0.9-, 0.8-, 0.7-, 0.6-, 0.5-, 0.4-, 0.3-, 0.2-, 0.1-, 0.05-or 0.01-fold or less of the reference level (e.g., predetermined from the reference sample).
In one embodiment of the invention, the biomarkers are measured separately. However, it is also contemplated that measurements may be taken together, i.e. the method of the invention may encompass the determination of more than one marker, i.e. two, three, four or five markers. If more than one biomarker is determined, the diagnostic algorithms for each marker are combined.
Preferred biomarker combinations are as follows:
GDF-15 and Urea
GDF-15 and SHBG
GDF-15 and PlGF
GDF-15 and IL-6
PlGF and IL-6
Troponin and sST2
hsCRP and sST2
PLGF and
In an embodiment of the method of the invention, the above-mentioned method further comprises the step (d) of recommending, starting or stopping a treatment comprising a statin.
The phrase "recommending a treatment" as used herein refers to using information or data generated relating to the level of at least one biomarker referred to herein in a patient sample to identify the patient as suitable or unsuitable for treatment with a treatment comprising statin. The phrase "recommending a treatment" as used herein may also refer to using the generated information or data for proposing or selecting a treatment that includes statin for a patient identified or selected as more likely or less likely to respond to a treatment that includes statin. The information or data used or generated may be in any form, written, spoken or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, sending, transmitting, providing, propagating, distributing, or a combination thereof. In some embodiments, the communicating, presenting, reporting, storing, sending, transmitting, providing, propagating, distributing, or a combination thereof is performed by a computer device, an analyzer unit, or a combination thereof. In some further embodiments, the communicating, presenting, reporting, storing, sending, transmitting, providing, propagating, distributing, or a combination thereof is performed by a laboratory or medical professional. In some embodiments, the information or data comprises a comparison of the level of at least one biomarker indicated herein to a reference level.
Preferably, if the patient is more likely to respond to a treatment comprising statin, the treatment comprising statin is initiated (in the case where the patient has not previously been treated with statin) or continued (in the case where the patient has not previously been treated with statin). Therefore, it is recommended to start or continue the treatment including statins.
Preferably, if the patient is less likely to respond to a treatment that includes statin, the treatment that includes statin is not initiated (in the case where the patient has not previously been treated with statin) or discontinued (in the case where the patient has not previously been treated with statin). Therefore, it is recommended that treatment including statins not be initiated or discontinued.
Accordingly, the present invention also relates to a method of treating a patient with heart failure, the method comprising:
a) measuring the level of at least one biomarker in a sample of the patient selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive c-reactive protein),
b) comparing the level of the at least one biomarker measured in a) with a respective reference level.
c) Identifying the patient as more or less likely to respond to a treatment comprising a statin, and optionally
d) Administering the statin or selecting a response comprising the statin to the patient when the level of the at least one biomarker is indicative of a response comprising the statin.
Treating heart failure by step d) mentioned above
In one embodiment, identifying the patient as more likely or less likely to respond to a treatment comprising statin as described in step c) is based on the comparison made in step b). Preferred diagnostic algorithms for individual markers are disclosed elsewhere herein. Depending on the biomarker, a level of the biomarker in the patient sample above or below the reference level is indicative for a patient more likely to respond to a treatment comprising statin (see the diagnostic algorithm described above). Step d) is performed if the level of the biomarker indicates a patient more likely to respond to a treatment comprising statin. If the level of the biomarker is indicative of a patient who is less likely to respond to a treatment comprising statin, step d) is not performed.
The definitions and explanations given hereinabove apply hereinafter (unless otherwise stated) mutatis mutandis.
Method for predicting the risk of patient mortality and/or hospitalization
The invention further relates to a method of predicting the risk of mortality and/or hospitalization of a patient, wherein said patient has heart failure and wherein said patient is undergoing a treatment comprising a statin, said method comprising:
(a) measuring in a sample of the patient the level of at least one marker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive c-reactive protein), and
(b) comparing the level of the at least one marker to a respective reference level.
The methods of the invention are preferably ex vivo or in vitro methods.
Preferably, the risk of the patient suffering from death or hospitalization is predicted based on step (b). Thus, the above-mentioned method may further comprise the step of predicting the risk of the patient suffering from death or hospitalization based on the result of step (b). In one embodiment, the method therefore further comprises the step (c) of predicting the risk of mortality and/or hospitalization of the patient, in particular when the level of the at least one biomarker in the patient sample is below or above a reference level (for diagnostic algorithms, see below).
In one embodiment, the level of at least one biomarker is measured by: binding the sample to a reagent that specifically binds to each label, thereby forming a complex between the reagent and the label, detecting the amount of complex formed, and thereby measuring the level of the label. This is particularly suitable if the biomarker to be measured is a polypeptide (GDF-15, SHBG, PLGF IL-6, transferrin, cardiac troponin, sFlt-1, prealbumin, ferritin, osteopontin, sST2 and hscRP). If the biomarker being measured is urea or uric acid, the level of said biomarker is measured by contacting the sample with an enzyme that catalyzes the conversion of said biomarker. Preferred enzymes are disclosed elsewhere herein.
The term "patient" has been defined above in the context of a method of identifying a subject as likely to respond to a treatment comprising a statin. Preferably, the patient being tested has heart failure as described above. Depending on the measured biomarker, the patient may additionally have or not have coronary artery disease (see above).
In addition, the patient should undergo a treatment comprising statins. Thus, the patient should have been treated with statin prior to obtaining the sample. In the context of the present invention, a patient who has been treated with statin prior to obtaining a sample should have been treated with statin prior to obtaining a sample, preferably for one month or more, more preferably for three months or more, or most preferably for six months or more.
The term "prediction" as used herein refers to assessing the likelihood that a patient will die and/or be hospitalized at a defined time window (prediction window) in view of this future. The prediction window is the interval in which the patient will die or be hospitalized according to the predicted likelihood. The prediction window may be the entire remaining life of the patient after analysis by the method of the invention. Preferably, however, the prediction window is an interval of 6 months, 12 months, 18 months, two years, three years or five years after the method of the invention has been carried out (more preferably and accurately, after the sample analysed by the method of the invention has been obtained). Most preferably, the prediction window is an interval of 18 months. As will be appreciated by those skilled in the art, such evaluations are generally not intended to be correct for 100% of the patients analyzed. However, this term requires that the assessment will be valid for a statistically significant portion of the patient being analyzed. Whether a moiety is statistically significant can be determined by one skilled in the art without further trouble using various well-known statistical evaluation tools, such as determination of 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, 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the possibilities envisaged by the present invention allow the prediction to be correct for at least 60%, at least 70%, at least 80% or at least 90% of the patients of a given population.
The term "death" as used herein preferably relates to death of any cause, and more preferably to death due to cardiac causes, and most preferably to death due to cardiovascular events. The term "hospitalization" as used herein preferably relates to hospitalization for any reason, and more preferably, to hospitalization for cardiac reasons, and most preferably to hospitalization for cardiovascular events. The term "cardiovascular event" as used herein refers to any condition of the cardiovascular system, including preferably any acute cardiovascular event. Preferably, the term also includes heart failure. The acute cardiovascular event is, preferably, Stable Angina Pectoris (SAP) or polar coronary syndrome (ACS). ACS patients may exhibit Unstable Angina (UAP) or Myocardial Infarction (MI). MI may be ST elevated MI (STEMI) or non-ST elevated MI (NSTEMI).
NSTE-ACS as used herein encompasses UAP and NSTEMI. The occurrence of MI may be followed by Left Ventricular Dysfunction (LVD), development of heart failure, or even death. Further preferred cardiovascular events encompass bradyarrhythmias or tachyarrhythmias including sudden cardiac death and stroke (cardiovascular event or accident). Furthermore, mortality may also refer to the mortality or the proportion of the number of deaths to a given patient population.
The expression "predicting the risk of mortality and/or hospitalization" as used herein means that the patients analyzed by the method of the invention are assigned to a group of patients with an increased risk population or a group with a decreased risk. An elevated risk according to the present invention preferably means that the risk of hospitalization or of death of the patient is significantly elevated (i.e. significantly increased) in a predetermined prediction window relative to the average risk of hospitalization or of death of the patient population. The reduced risk referred to according to the present invention preferably means that the risk of hospitalization or of death of the patient within a predetermined prediction window is significantly reduced with respect to the average risk of hospitalization or of death of the patient population. In particular, a significant increase or decrease in risk is an increase or decrease in risk to the extent that it is considered to be meaningful for prognosis, in particular the increase or decrease is considered to be statistically significant. The terms "significant" and "statistically significant" are known to those skilled in the art. Thus, whether an increase or decrease in risk is significant or statistically significant can be determined without further trouble by one of skill in the art using a variety of well-known statistical evaluation tools.
Preferably, for a prediction window of 18 months, an elevated and thus increased risk of mortality and/or hospitalization using herein preferably relates to an increased risk of more than 20%, or more preferably, more than 25%, and most preferably, more than 30%. A reduction of the risk of mortality or hospitalization as used herein preferably relates to a risk reduction of more than 10%, more preferably more than 15%, most preferably more than 20%, preferably with respect to a prediction window of 18 months (compared to the average risk, see paragraph above).
The term "reference level" has been defined above. The definition is applied accordingly. Thus, the term preferably refers to a level that allows assigning patients to a group of patients with an increased risk of mortality and/or hospitalization, or a group of patients with a reduced risk of mortality and/or hospitalization. Such a reference level may be a threshold level separating each of these groups. Thus, the reference level of a biomarker referred to herein should be a level that allows assigning a patient to a group of patients with an increased risk of mortality and/or hospitalization, or a group of patients with a reduced risk of mortality and/or hospitalization. Suitable threshold levels for separating the two groups may be calculated without further trouble by statistical tests referred to elsewhere herein based on the levels of the biomarkers referred to herein from patients (or from groups of such patients) having an increased risk of mortality and/or hospitalization or patients (or from groups of such patients) having a reduced risk of mortality and/or hospitalization. Preferred reference levels that may be derived from a patient or group of patients as mentioned above are indicated elsewhere herein.
Osteopontin as biomarker
If osteopontin is used as the biomarker, the patient being tested preferably does not suffer from coronary artery disease. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker below a reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
sST2 as biomarker
If sST2 is used as biomarker, the patient tested preferably does not suffer from coronary artery disease. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
GDF-15 as biomarker
If GDF-15 is used as a biomarker, the patient being tested may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
Urea as biomarker
If urea is used as the biomarker, the patient being tested may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
Uric acid as biomarker
If uric acid is used as a biomarker, the patient being tested may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
Transferrin as a biomarker
If transferrin is used as a biomarker, the patient being tested preferably also suffers from coronary artery disease. Preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
Cardiac troponin as biomarker
If cardiac troponins, in particular troponin T, are used as biomarkers, the patient to be tested may or may not additionally suffer from coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
SHBG as biomarker
If SHBG is used as the biomarker, the patient being tested may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
sFlt-1 as biomarker
If sFlt-1 is used as a biomarker, the patient being tested may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
Prealbumin as a biomarker
If prealbumin is used as the biomarker, the patient being tested may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
PlGF as biomarkers
If PlGF is used as a biomarker, the patient being tested may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
IL-6 as biomarker
If IL-6 is used as a biomarker, the patient being tested may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
Ferritin as a biomarker
If ferritin is used as a biomarker, the patient being tested preferably also suffers from coronary artery disease. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
HsCRP as biomarker
If hsCRP is used as the biomarker, the patient being tested may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
If the patient suffers from coronary artery disease, the following applies: preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
Preferred reference levels are disclosed in the context of a method of identifying a patient as susceptible to treatment comprising a statin. Preferred diagnostic algorithms for each marker are also disclosed in the section "preferred embodiments", see, e.g., embodiment 10.
The invention also relates to i) at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein) and/or ii) a binding agent that specifically binds a biomarker selected from the group consisting of: use of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein), or iii) an enzyme or compound that allows the conversion of uric acid or urea for the identification of a patient as likely to respond to a treatment comprising inhibin.
The invention also relates to i) at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein) and/or ii) a binding agent that specifically binds a biomarker selected from the group consisting of: use of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein), or iii) an enzyme or compound that allows the conversion of uric acid or urea for predicting the risk of death and/or hospitalization of said patient.
The invention also relates to i) at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein) and/or ii) a binding agent that specifically binds a biomarker selected from the group consisting of: use of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein), or iii) an enzyme or compound that allows the conversion of uric acid or urea in the preparation of a medicament or diagnostic composition for identifying a patient with heart failure as likely to respond to a treatment comprising inhibin.
The invention also relates to i) at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein) and/or ii) a binding agent that specifically binds a biomarker selected from the group consisting of: use of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein), or iii) an enzyme or compound that allows the conversion of uric acid or urea for the preparation of a medicament or diagnostic composition for predicting the risk of mortality and/or hospitalization of patients with heart failure and undergoing treatment including statins.
According to a preferred embodiment of the invention, there is provided an apparatus adapted to carry out the method of the invention, comprising:
a) an analyzer unit comprising at least one binding agent that specifically binds to at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein), or an enzyme or compound that allows for uric acid or urea conversion, said unit being adapted to measure the level of one or more biomarkers in a sample of a patient with heart failure, and
b) an analyzer unit for comparing the determined levels with reference levels, whereby the patient is identified as more or less likely to respond to a treatment comprising statin, said unit comprising a database with one or more reference levels and a computer-implemented algorithm for carrying out the comparison.
According to another preferred embodiment of the invention, there is provided an apparatus adapted to carry out the method of the invention, comprising:
a) an analyzer unit comprising one or more binding agents that specifically bind to at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein), or iii) an enzyme or compound that allows uric acid or urea conversion, said unit being adapted to measure the level of one or more biomarkers in a sample of a patient with heart failure, and
b) an analyzer unit for comparing the determined levels with reference levels, the unit comprising a database with one or more reference levels and a computer-implemented algorithm for performing the comparison, thereby predicting the risk of patient mortality and/or hospitalization.
Preferred reference levels and algorithms are disclosed elsewhere herein.
Preferred embodiments of the present disclosure include systems for identifying a patient as likely to respond to a therapy comprising a statin. Examples of systems include clinical chemistry analyzers, coagulation chemistry analyzers, immunochemistry analyzers, urine analyzers, nucleic acid analyzers for detecting the results of or monitoring the progress of a chemical or biological reaction. More specifically, an exemplary system of the present disclosure may include Roche ElecsysTMSystems and Cobas®e immunoassay analyzer, Abbott ArchitectTMAnd AxsymTMAnalyzer, Siemens CentaurTMAnd ImmuliteTMAnalyzer and Beckman Coulter UniCelTMAnd AccessTMAn analyzer, and the like.
Embodiments of the system may include one or more analyzer units for practicing the present disclosure. The analyzer unit of the system disclosed herein is in operable communication with the computing device disclosed herein via any known wired connection, bluetooth, LANS, or wireless signal. Additionally, according to the present disclosure, the analyzer unit comprises a stand-alone device, or a module in a larger instrument, which performs one or both of the assays, e.g., qualitative and/or quantitative evaluation of the sample for diagnostic purposes. For example, the analyzer unit may perform or assist with aspiration, dosing, mixing of the sample and/or reagents. The analyzer unit may comprise a reagent fixing unit for fixing a reagent to perform the assay. The reagents may be arranged, for example, in the form of containers or cassettes containing individual reagents or groups of reagents, arranged in suitable containers (receptacle) or locations in a storage compartment or conveyor. The detection reagent may also be in an immobilized form on a solid support, which is contacted with the sample. Further, the analyzer unit may comprise processing and/or detection components optimized for a specific analysis.
According to some embodiments, the analyzer unit may be configured for optical detection of analytes, such as labels, with the sample. Exemplary analyzer units configured for optical detection include devices configured to convert electromagnetic energy into electrical signals, including single-element and multi-element or array optical detectors. In accordance with the present disclosure, an optical detector is capable of monitoring the photo-magnetic signal and providing an electrical output signal or a response signal relative to a baseline signal that is indicative of the presence and/or concentration of an analyte in a sample located in the optical path. Such devices may also include, for example, photodiodes, including avalanche photodiodes, phototransistors, photoconductive detectors, linear sensor arrays, CCD detectors, CMOS detectors, including CMOS array detectors, photomultiplier tubes, and photomultiplier tube arrays. According to certain embodiments, the optical detector, such as a photodiode or photomultiplier tube, may contain additional signal conditioning or processing electronics. For example, the optical detector may comprise at least one preamplifier, electronic filter or integrated circuit. Suitable preamplifiers include, for example, integrated, transimpedance and current gain (current mirror) preamplifiers.
Additionally, one or more analyzer units according to the present disclosure may include a light source for emitting light. For example, the light source of the analyzer may consist of at least one light emitting element (such as a light emitting diode, an electrokinetic radiation source such as an incandescent lamp, an ionizing light emitting lamp, a gas discharge lamp, a high intensity gas discharge lamp, a laser) for measuring the analyte concentration of the sample being tested or for enabling energy transfer (e.g., by fluorescence resonance energy transfer or catalytic enzymes).
In addition, the analyzer unit of the system may include one or more incubation units (e.g., for maintaining the sample or reagent at a particular temperature or temperature range). In some embodiments, the analyzer unit may comprise a thermal cycler, including a real-time thermal cycler, for subjecting the sample to repeated temperature cycles and monitoring the sample for changes in the level of amplification products.
Additionally, the analyzer unit of the systems disclosed herein may include or be operatively connected to a reaction vessel or a cuvette feeding unit. Exemplary priming units include liquid handling units, such as pipetting units, to deliver samples and/or reagents to reaction vessels. The aspiration unit may comprise a reusable washable needle, e.g. a steel needle or a disposable tip. The analyzer unit may further comprise one or more mixing units, such as a vibrator to vibrate the cuvette containing the liquid, or a paddle to mix the liquid in the cuvette or reagent container.
Following the above, portions of some steps of the methods disclosed and described herein may be performed by a computing device, according to some embodiments of the present disclosure. The computing device may be, for example, a general purpose computer or a portable computing device. It should be understood that multiple computing devices may be used together, such as over a network or other data transmission method, for performing one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants ("PDAs"), such as BLACKBERRY brand devices, mobile devices, tablets, servers, and the like. Typically, a computing device includes a processor capable of executing a variety of instructions (such as a software program).
The computing device accesses the memory. The memory is a computer-readable medium and may include a single storage device or multiple storage devices, which may be located locally to the computing device or may be connected to the computing device over a network, for example. Computer readable media can be any available media that can be accessed by a computing device and includes both volatile and nonvolatile media. Further, the computer readable medium may be one or both of a removable medium and a non-removable medium. By way of example, and not limitation, computer readable media may comprise computer storage media. Exemplary computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory or any other memory technology, CD-ROM, Digital Versatile Disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store a plurality of instructions that can be accessed by a computing device and executed by a processor of the computing device.
According to embodiments of the present disclosure, software may include instructions that, when executed by a processor of a computing device, may perform one or more steps of the methods disclosed herein. Some instructions may be adapted to generate signals that control the operation of other machines and may therefore be operated by those control signals to convert material remote from the computer itself. These descriptions and representations are the means used by those skilled in the data processing arts to most effectively convey the substance of their work to others skilled in the art, for example.
The plurality of instructions may also include an algorithm that is generally conceived to be a self-consistent sequence of steps leading to a desired result. The steps are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic pulses or signals capable of being stored, transferred, transformed, combined, compared, and otherwise manipulated. It has proven convenient at times, principally for reasons of common usage, to refer to these signals as values, features, display data, quantities, or the like, when referring to the physical items or representations in which such signals are embodied or expressed. It should be borne in mind, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities. According to some embodiments of the present disclosure, an algorithm for performing a comparison between a determined level of one or more markers disclosed herein and a suitable reference is embodied by executing instructions and performed. These results can be given as the output of raw data for parametric diagnosis or as absolute or relative levels. According to various embodiments of the systems disclosed herein, "diagnosis" may be provided by a computing device of the systems disclosed herein based on the comparison of the calculated "level" to a reference or threshold. For example, the computing device of the system may provide an indicator in the form of text, symbols, or numerical values that indicate a particular diagnosis.
The computing device may also access an output device. Exemplary output devices include, for example, facsimile machines, displays, printers, and documents. According to some embodiments of the present disclosure, a computing device may perform one or more steps of the methods disclosed herein, and then provide an output via an output device regarding the results, indications, proportions or other factors of the methods.
Finally, the invention relates to a kit suitable for performing the method of the invention, said kit comprising a binding agent that specifically binds to at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (high sensitive C-reactive protein) reference standards, as well as instructions for carrying out said method.
The term "kit" as used herein refers to a collection of the above-mentioned components, preferably provided separately or in a single container. The container also contains instructions for carrying out the method of the invention. These instructions may be provided in the form of a manual or may be provided by computer program code (which is capable of performing the comparisons relating to the methods of the invention and thus establishing a diagnosis when implemented in a computer or data processing apparatus). The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g. an optical disc), or directly on a computer or data processing device. Furthermore, the kit should comprise at least one standard for the above-defined reference, i.e. a solution with a predetermined level of the biomarker as mentioned herein representative of the reference level.
In some embodiments, the kits disclosed herein comprise at least one component or a combination of component packages for practicing the disclosed methods. By "packaged combination" is meant that the kit provides a single package containing a combination of one or more components, such as probes (e.g., antibodies), controls, buffers, instructions for reagents (e.g., conjugates and/or substrates), and the like, as disclosed herein. Kits containing a single container are also included in the definition of "packaged combination". In some embodiments, the kit comprises at least one probe, such as an antibody (with a particular affinity for an epitope of a biomarker disclosed herein). For example, the kit may include an antibody labeled with fluorescein or an antibody that is a member of a fusion protein. In the kit, the probe may be immobilized, and may be immobilized in a specific conformation. For example, immobilized probes can be provided in a kit to specifically bind to a target protein, to detect the target protein in a sample, and/or to remove the target protein from a sample.
According to some embodiments, the kit comprises at least one probe, which may be immobilized, in at least one container. The kit may also comprise a plurality of probes, preferably immobilized, in one or more containers. For example, multiple probes may be present in a single container, or in separate containers, e.g., where each container contains a single probe.
In some embodiments, a kit may include one or more non-immobilized probes and one or more solid supports, which may or may not include immobilized probes. Some such embodiments may include some or all of the reagents or supplies required for immobilizing one or more probes on a solid support, or for binding immobilized probes to some or all of a particular protein in a sample.
In certain embodiments, a single probe (which includes multiple copies of the same probe) may be immobilized to a single solid support and provided in a single container. In other embodiments, two or more probes, each specific for a different target protein or a different form of a single target protein (such as a particular epitope), are provided in a single container. In some such embodiments, the immobilized probes can be provided in a plurality of different containers (e.g., single use format), or a plurality of immobilized probes can be provided in a plurality of different containers. In further embodiments, the probes may be immobilized on a plurality of different types of solid supports. Any combination of immobilized probes and containers is contemplated for the kits disclosed herein, and any combination thereof may be selected to achieve a suitable kit for a desired use.
The container of the kit can be any container suitable for packaging and/or containing one or more components disclosed herein, including, for example, probes (e.g., antibodies), controls, buffers, and reagents (e.g., conjugates and/or substrates). Suitable materials include, but are not limited to, glass, plastic, cardboard or other paper products, wood, metal, or any alloy thereof. In some embodiments, the container may completely surround the immobilized probe or may simply cover the probe to minimize contamination and exposure to light due to dust, oil, and the like. In some further embodiments, a kit may comprise a single container or a plurality of containers, and when a plurality of containers is present, each container may be the same as all other containers, different from other containers, or different from some but not all other containers.
Furthermore, the present invention relates to a statin for use in the treatment of heart failure in a patient having a level (preferably in a sample, more preferably in a blood, serum or plasma sample, most preferably in a plasma sample) of such at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (high sensitive C-reactive protein), which level is higher or lower than a respective reference level.
Finally, the invention relates to the use of a statin for the preparation of a medicament for the treatment of heart failure in a patient, the patient has a level (preferably in a sample, more preferably in a blood, serum or plasma sample, most preferably in a plasma sample) of at least one biomarker selected from the group consisting of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein), which is higher or lower than a respective reference level (of the respective marker).
In one embodiment, the level is a blood, serum or plasma level, in particular a plasma level.
Furthermore, as noted above, the patient may additionally suffer from coronary artery disease, or may not suffer from coronary artery disease.
Preferred reference levels are disclosed elsewhere herein.
Osteopontin as biomarker
If the biomarker is osteopontin, the patient preferably does not suffer from coronary artery disease. Preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
sST2 as biomarker
If the biomarker is sST2, the patient preferably does not have coronary artery disease. Preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
GDF-15 as biomarker
If the biomarker is GDF-15, the patient may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is higher than the biomarker reference level.
If the patient suffers from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
Urea as biomarker
If the biomarker is urea, the patient may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, the patient being treated has a biomarker level that is higher than the biomarker reference level.
Uric acid as biomarker
If the biomarker is uric acid, the patient may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
Transferrin as a biomarker
If the biomarker is transferrin, the patient preferably also suffers from coronary artery disease. Preferably, the patient being treated has a biomarker level that is higher than the biomarker reference level.
Cardiac troponin as biomarker
If the biomarker is cardiac troponin, in particular troponin T, the patient may or may not additionally suffer from coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
If the patient suffers from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is higher than the biomarker reference level.
SHBG as biomarker
If the biomarker is SHBG, the patient may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
If the patient suffers from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is higher than the biomarker reference level.
sFlt-1 as biomarker
If the biomarker is sFlt-1, the patient may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
Prealbumin as a biomarker
If the biomarker is prealbumin, the patient may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is higher than the biomarker reference level.
If the patient suffers from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
PlGF as biomarkers
If the biomarker is PlGF, the patient may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization. Also preferably, a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization.
IL-6 as biomarker
If the biomarker is IL-6, the patient may or may not have coronary artery disease. However, it is particularly envisaged that the patient suffers from CAD. Preferably, the patient being treated has a biomarker level that is higher than the biomarker reference level.
Ferritin as a biomarker
If the biomarker is ferritin, the patient preferably also suffers from coronary artery disease. Preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
HsCRP as biomarker
If the biomarker is hsCRP, the patient may or may not have coronary artery disease.
If the patient does not suffer from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is higher than the biomarker reference level.
If the patient suffers from coronary artery disease, the following applies: preferably, the patient being treated has a biomarker level that is lower than the biomarker reference level.
Preferred reference levels are indicated above, along with methods of identifying a patient as likely to respond to a treatment comprising a statin. Preferred diagnostic algorithms for individual markers are also disclosed in the following section of "preferred embodiments", see embodiment 16.
Preferred embodiments
Preferred embodiments of the invention are disclosed below. The definitions given above apply mutatis mutandis.
1. A method of identifying a patient with heart failure as likely to respond to a treatment comprising a statin, comprising:
(a) measuring the level of at least one biomarker in a sample of the patient selected from the group consisting of: GDF-15 (growth differentiation factor 15), SHBG (sex hormone binding globulin), PLGF (placental growth factor), IL-6 (interleukin-6), urea, uric acid, transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive c-reactive protein), and
(b) comparing the level of the at least one marker to a respective reference level.
2. The method according to
3. The method of
4. The method of
5. The method of any one of claims 1-4, wherein the statin is selected from the group consisting of: atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin.
6. The method of any one of embodiments 1-5, wherein the patient has been treated with statin prior to obtaining the sample.
7. The method of any one of embodiments 1-6, wherein the patient has not been treated with statin prior to obtaining the sample.
8. The method of any one of embodiments 1-7, wherein
i) At least one biomarker is GDF-15, and
a) wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is lower than a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
ii) at least one biomarker is SHBG, and
a) wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient sample that is lower than a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than a reference level indicates that the patient is less likely to respond to a treatment comprising statin, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
iii) the at least one biomarker is PlGF, and wherein a level of the biomarker in the patient's sample that is greater than a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient's sample that is less than the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
iv) the at least one biomarker is IL-6, and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
v) the at least one biomarker is urea, and wherein a level of the biomarker in the patient sample above a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample below a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
vi) the at least one biomarker is osteopontin, and wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient's sample below a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient's sample above a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
vii) the at least one biomarker is sST2, and wherein the patient does not suffer from coronary artery disease, and wherein a level of the biomarker in the patient's sample that is below the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient's sample that is above the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
viii) at least one biomarker is uric acid, and wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
ix) at least one biomarker is sFlt-1, and wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
x) at least one biomarker is transferrin, and wherein the patient also suffers from coronary artery disease, and wherein a level of the biomarker in the patient's sample above a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient's sample below a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
xi) at least one biomarker is ferritin, and wherein the patient further suffers from coronary artery disease, and wherein a level of the biomarker in the patient's sample below a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient's sample above a reference level indicates that the patient is less likely to respond to a treatment comprising statin,
xii) at least one biomarker is cardiac troponin, and
a) wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient sample that is lower than a reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than a reference level indicates that the patient is less likely to respond to a treatment comprising statin, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin,
and/or
xiii) wherein at least one biomarker is prealbumin and/or hsCRP, and
a) wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is less likely to respond to a treatment comprising statin, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient is more likely to respond to a treatment comprising statin, and/or wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient is less likely to respond to a treatment comprising statin.
9. A method of predicting the risk of mortality and/or hospitalization of a patient, wherein said patient has heart failure and wherein said patient is undergoing treatment comprising a statin, said method comprising:
(a) measuring in a sample of said patient the level of at least one marker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive c-reactive protein), and
(b) comparing the level of the at least one marker to a respective reference level.
10. The method according to embodiment 9, wherein
i) At least one biomarker is GDF-15, and
a) wherein the patient does not have coronary artery disease and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
ii) at least one biomarker is SHBG, and
a) wherein the patient does not have coronary artery disease and wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
iii) the at least one biomarker is PlGF, and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
iv) the at least one biomarker is IL-6, and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
v) the at least one biomarker is urea, and wherein a level of the biomarker in the patient sample above the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample below the reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
vi) the at least one biomarker is osteopontin, and wherein the patient does not have coronary artery disease, and wherein a level of the biomarker in the patient sample that is below the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is above the reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
vii) at least one biomarker is sST2, and wherein the patient does not suffer from coronary artery disease, and wherein a level of the biomarker in the patient sample below a reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample above a reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
viii) at least one biomarker is uric acid, and wherein a level of the biomarker in the patient sample below a reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample above the reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
ix) at least one biomarker is sFlt-1, and wherein a level of the biomarker in the patient sample below a reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample above the reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
x) the at least one biomarker is transferrin, and wherein the patient further suffers from coronary artery disease, and wherein a level of the biomarker in the patient sample above a reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample below a reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
xi) the at least one biomarker is ferritin, and wherein the patient further suffers from coronary artery disease, and wherein a level of the biomarker in the patient sample below a reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample above a reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
xii) at least one biomarker is cardiac troponin, and
a) wherein the patient does not have coronary artery disease and wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization,
and/or
xiii) at least one biomarker is prealbumin and/or hsCRP, and
a) wherein the patient does not have coronary artery disease and wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization, or
b) Wherein the patient has coronary artery disease and wherein a level of the biomarker in the patient sample that is lower than the reference level indicates that the patient has a reduced risk of mortality and/or hospitalization, and/or wherein a level of the biomarker in the patient sample that is higher than the reference level indicates that the patient has an increased risk of mortality and/or hospitalization.
11. The method according to any one of embodiments 1-10, wherein the sample is a blood, serum or plasma sample.
12. The method according to any one of embodiments 1-11, wherein the patient is a human.
13. In a sample of a patient with heart failure i) at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein) and/or ii) at least one binding agent that specifically binds a biomarker selected from the group consisting of: use of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein) for identifying a patient as likely to respond to a treatment comprising a statin.
14. In a sample of a patient with heart failure i) at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein) and/or ii) at least one binding agent that specifically binds a biomarker selected from the group consisting of: use of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hsCRP (hypersensitive C-reactive protein) for predicting the risk of mortality and/or hospitalization of the patient.
15. Apparatus for carrying out the method according to any one of
a) An analyzer unit comprising at least one binding agent that specifically binds to at least one biomarker selected from the group consisting of: GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (hypersensitive C-reactive protein), said unit being adapted to measure the level of one or more biomarkers in a sample of a patient with heart failure, and
b) an analyzer unit for comparing the determined level with a reference level, whereby the patient is identified as more or less likely to respond to a treatment comprising inhibin, said unit comprising a database with one or more reference levels, and a computer-implemented diagnostic algorithm for performing the comparison, in particular wherein the diagnostic algorithm is an algorithm as claimed in claim 8.
16. Inhibin for use in the treatment of heart failure in a patient having such a level of at least one biomarker, in particular a blood, serum or plasma level, selected from the group consisting of GDF-15 (growth differentiation factor 15), urea, SHBG (sex hormone binding globulin), uric acid, PLGF (placental growth factor), IL-6 (interleukin-6), transferrin, cardiac troponin, sFlt-1 (soluble fms-like tyrosine kinase-1), prealbumin, ferritin, osteopontin, sST2 (soluble ST2) and hscRP (high sensitive C-reactive protein), which is higher or lower than a respective reference level, in particular wherein
i) At least one biomarker is GDF-15, and
a) wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is higher than a reference level, or
b) Wherein the patient has coronary artery disease, and wherein the level of the biomarker is lower than a reference level,
ii) at least one biomarker is SHBG, and
a) wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is lower than a reference level, or
b) Wherein the patient has coronary artery disease, and wherein the level of the biomarker is higher than a reference level,
iii) at least one biomarker is PlGF, and wherein the level of said biomarker is higher than a reference level,
iv) at least one biomarker is IL-6, and wherein the level of said biomarker is higher than a reference level,
v) at least one biomarker is urea, and wherein the level of said biomarker is higher than a reference level,
vi) at least one biomarker is osteopontin, and wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is lower than a reference level,
vii) at least one biomarker is sST2, and wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is lower than a reference level,
viii) at least one biomarker is uric acid, and wherein the level of said biomarker is lower than a reference level,
ix) at least one biomarker is sFlt-1, and wherein the level of said biomarker is lower than a reference level,
x) at least one biomarker is transferrin, and wherein the patient also suffers from coronary artery disease, and wherein the level of the biomarker is higher than a reference level,
xi) at least one biomarker is ferritin, and wherein the patient also suffers from coronary artery disease, and wherein the level of the biomarker is lower than a reference level,
xii) at least one biomarker is cardiac troponin, and
a) wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is lower than a reference level, or
b) Wherein the patient has coronary artery disease, and wherein the level of the biomarker is higher than a reference level,
and/or
xiii) at least one biomarker is prealbumin and/or hsCRP, and
a) wherein the patient does not suffer from coronary artery disease, and wherein the level of the biomarker is higher than a reference level, or
b) Wherein the patient has coronary artery disease, and wherein the level of the biomarker is lower than a reference level.
All references cited in this specification are incorporated herein by reference for their entire disclosure and for the disclosure specifically mentioned in this specification.
Drawings
Figure 1 Kaplan-Meier curves of (split) time separated by CAD, statin treatment (0 = no statin, n =211, 1= at statin treatment, n = 288), and median biomarker level at baseline (0 = below median, 1= above median; occasionally 1= below median, 2= above median) versus first HF hospitalization or death; CAD =0 patient does not have (w/o) coronary artery disease, CAD =1, patient has coronary artery disease.
Detailed Description
The following examples will illustrate the invention only. They should not be construed in any way as limiting the scope of the invention.