sign in sign up
<<
Home , Anitrazafen, Bendazac, Flumizole

(19) &  

(11) EP 2 133 696 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 16.12.2009 Bulletin 2009/51 G01N 33/68 (2006.01)

(21) Application number: 08010524.0

(22) Date of filing: 10.06.2008

(84) Designated Contracting States: (72) Inventor: Spanuth, Eberhardt AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 69221 Dossenheim (DE) HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR (74) Representative: Prüfer & Partner GbR Designated Extension States: European Patent Attorneys AL BA MK RS Sohnckestrasse 12 81479 München (DE) (71) Applicant: Spanuth, Eberhardt 69221 Dossenheim (DE)

(54) Very low concentrations of troponin I or T for assessing a cardiovascular risk with respect to the administration of anti-inflammatory drugs

(57) The present invention relates to a method for ponin I or T diagnosing the risk of a patient to suffer from a cardio- b) diagnosing the risk of the patient by comparing the vascular complication as a consequence of administra- measured level to known leves associate with different tion of a COX-2 inhibiting compound, comprising the grades of risk in a patient. steps of a) measuring the level of very low concentrations of tro- EP 2 133 696 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 133 696 A1 2

Description Mukherjee D, Nissen SE, Topol EJ. Risk of cardiovascu- lar events associated with selective COX-2 Inhibitors. JA- [0001] The present invention relates to the use of very MA 2001; 286: 954-959.). low concentrations of troponin I or T for assessing the [0006] Cardiovascular complications, such as acute cardiovascular risk of a patient with respect to the admin- 5 myocardial infarction or stroke, may lead to serious istration of an anti-inflammatory drug, particularly health problems and even death. Therefore, many pa- NSAIDs (non-steroidal anti-inflammatory drugs) or ster- tients who would strongly benefit from treatment with se- oids, more particularly selective Cox-2 inhibitors. lective Cox-2 inhibitors are not treated as it is unknown [0002] An aim of modem medicine is to provide per- whether treatment may result in a cardiovascular com- sonalized or individualized treatment regimens. Those 10 plication. are treatment regimens which take into account a pa- [0007] Cardiovascular problems or risks can remain tient’s individual needs or risks. Of particular importance asymptomatic for long periods of time. Therefore, reliable is the cardiovascular risk or complication, particularly an diagnosis of the presence of a cardiovascular risk is more unrecognized cardiovascular risk or complication. difficult and error-prone than generally believed. In par- [0003] Selective Cox-2 inhibitors are widely used po- 15 ticular, general practitioners and non-cardiologists often tent anti-inflammatory drugs. Compared to many other are not able to identify a previously unrecognized cardi- anti-inflammatory drugs, they appear to cause less gas- ovascular problem. trointestinal side-effects, such as gastrointestinal bleeds [0008] According to the state of the art, it is only pos- or ulcers. Therefore, for many patients treatment with sible to exclude patients with cardiovascular symptoms selective Cox-2 inhibitors is the treatment of choice. 20 or a known history of heart disease or hypertension from [0004] However, it has been noted, that selective Cox- treatment with selective Cox-2 inhibitors. 2 inhibitors can lead to cardiovascular complications, [0009] This risk management is insufficient, as also possibly followed by cardiac decompensation and even asymptomatic patients may develop a cardiovascular death. (VIOXX™), a selective Cox-2 inhibitor, complication due to an unrecognized predisposition, e.g. was removed from the market voluntarily by the manu- 25 the presence of an arterial plaque. As mentioned earlier, facturing company, Merck, after a 3.9 times rise in the only patients without any recognisable cardiovascular rate of serious thromboembolic incidents had led to pre- risk were included in the APPROVE study, yet treatment mature discontinuation of the APPROVE study (Adeno- led to cardiovascular complications in several patients. matous Polyp Prevention On Vioxx). Only patients with- [0010] This does not only apply with respect to selec- out any recognisable cardiovascular risk were included 30 tive Cox-2 inhibitors, but also with respect to other class- in that study for the secondary prevention of colon ade- es of anti-inflammatory drugs, e.g. being inhibitors of nomas. Even in the early post-observation phase higher Cox-2 and other target (e.g. Cox-1), and which may blood pressure levels had been noticed with 25 mg ro- cause cardiovascular complications. Examples for these fecoxib than with a placebo. 16 additional cardiovascular other classes of anti-inflammatory drugs include non-se- incidents per 1,000 treated patients (myocardial infarc- 35 lective Cox-2 inhibitors (compounds inhibiting Cox-1 and tion, stroke) with 25 mg rofecoxib compared with a pla- Cox-2). Even though the risk of leading to cardiovascular cebo (Topol EJ. Failing the public health--rofecoxib, Mer- complications is not as high as for selective Cox-2 inhib- ck, and the FDA. N Engl J Med 2004; 351: 1707-9) were itors, there may be cases where a possible cardiovascu- noticed after 18 months of follow-up. An increased car- lar complication has to be taken into account. diovascular risk has also been suspected in other studies 40 [0011] For example, cardiovascular side-effects are al- (VIGOR) and has been supported by data from a recent so suspected for other anti-inflammatory drugs, in par- meta-analysis (Bombardier C, Laine L, Reicin A, Shapiro ticular other NSAIDs or steroids. D, Burgos-Vargas R, Davis B, Day R, Ferraz MB, Hawkey [0012] WO 2006/077265 discloses a method for diag- CJ, Hochberg MC, Kvien TK, Schnitzer TJ; VIGOR Study nosing the risk of a patient to suffer from a cardiovascular Group. Comparison of upper gastrointestinal toxicity of 45 complication as a consequence of administration of a rofecoxib and in patients with rheumatoid ar- Cox-2 inhibiting compound, comprising the steps of a) thritis. VIGOR Study Group. N Engl J Med 2000; 343: measuring the level of a cardiac hormone, b) diagnosing 1520-8; Jüni P, Nartey L, Reichenbach S, Sterchi R, Di- the risk of the patient by comparing the measured level eppe PA, Egger M. Risk of cardiovascular events and to known levels associated with different grades of risk rofecoxib: cumulative meta-analysis. Lancet 2004; online 50 in a patient. ahead). [0013] In some cases, so-called molecular markers [0005] Similar cardiovascular side-effects are sus- permit to establish rapid and sufficiently accurate diag- pected within the scope of a class effect for other selec- nosis of the pathological state of a subject. A prominent tive (COX)-2 inhibitors such as celecox- example is troponin T and/or troponin I for the diagnosis ib (Celebrex™, Pfizer), (Prexige™, Novartis) 55 of MI, as mentioned beforehand, or natriuretic peptides, and , a pro-drug of , although at a in particular BNP and NT-proBNP for various non- lower incidence rate (Fitzgerald GA. Coxibs and cardio- ischemic heart diseases, e.g. heart failure. Blood levels vascular disease. N Engl J Med 2004; 351: 1709-1711. of cardiac troponins are sensitive and specific markers

2 3 EP 2 133 696 A1 4 of myocardial injury that are routinely used for the diag- In particular, the diagnostic means should be reliable and nosis of acute myocardial infarction. With regard for the suited for use by general practitioners and non-cardiol- determination of troponin, as an example of an ischemic ogists. marker, reference is made to Katus et al. (Mol Cell Cardiol [0016] The object of the invention is attained by a meth- 1989; 21:1349-53), Hamm et al. (NEJM 1992; 327: 5 od for diagnosing the risk of a patient to suffer from a 146-50), Ohmann et al. (NEJM 1996; 335: 1333-34), cardiovascular complication ("cardiovascular risk") as a Christenson et al. (ClinChem 1998; 44: 494-501), and to consequence of administration of an anti-inflammatory EP-A-0 394 819. Particularly preferred test for detection drug, particularly an NSAID, steroid, or a selective Cox- of troponin T are elektrochemiluminescence immu- 2 inhibitor, comprising the steps of noassays and for detection of troponin I fluorescence po- 10 larization immunoassays, respectively, reference is a) measuring the level of Very low concentrations of made to James et al. (ClinChem 2006; 52: 6832-37), and troponin I or T, numerous other publications. Troponin levels may also be elevated in some patients b) diagnosing the risk of the patient by comparing with heart failure, but blood levels are shown to be much 15 the measured level to known levels associated with lower compared with patients with acute myocardial inf- different grades of risk in a patient. arction when currently available assays are used. [0014] Recently, previously undetectable very low [0017] The object of the invention is furthermore at- concentrations of troponin I measured by the second- tained by a method for diagnosing the cardiovascular risk generation Stratus CS® with reagents provided by Dade 20 of a patient who is a candidate for administration of a Behring suggest that troponin values below the 99th per- compound having Cox-2 inhibiting properties, compris- centile could contain diagnostic information in patients ing the steps of with heart diseases. The limit of detection for this assay is 0.02 Pg/L, the 99th percentile is 0.07 Pg/L, and the a) measuring, preferably in vitro, the level of Very lowest concentration with a CV <10% has been reported 25 low concentrations of troponin I or T, as both 0.06 Pg/L and 0.10 Pg/L, respectively. In addition, very low concentrations of troponin T measured by using b) diagnosing the risk of the patient by comparing a new high sensitive troponin T assay (hsTnT, Roche the measured level to known levels associated with Diagnostics Elecsis Troponin T 5th Generation) have different grades of risk in a patient. been suggested to be an indicator for adverse outcomes 30 in chronic heart failure (Latini et al. Circulation 2007; 116: [0018] The compound having Cox-2 inhibiting proper- 1242-49) In this issue of Circulation, Latini and col- ties (also referred to as Cox-2 inhibitor) can also be any leagues report results of an investigation in which they compound having anti-inflammatory properties (e.g. it measured circulating troponin T in 4053 patients with can be a steroid or NSAID). Most preferably, the com- heart failure using a high-sensitivity troponin (hsTnT) as- 35 pound is a selective Cox-2 inhibitor. say. Whereas circulating troponins were detectable in [0019] The object of the invention is also attained by 10% of the patients with heart failure when conventional a method of deciding on the possible treatment of a pa- assays were used, 90% of the patients had detectable tient with a compound having Cox-2 inhibiting properties, troponin T levels using the hsTnT assay. The authors which method comprises report that higher hsTnT was a marker of greater severity 40 of heart failure, was associated with greater risk of ad- a) measuring, preferably in vitro, the level of Very verse outcomes, and added incremental value to predic- low concentrations of troponin I or T in the patient, tion models for death. Similar results were obtained by b) comparing the measured level with known levels using new high sensitive troponin I fluorescence polari- associated with different grades of risk in a patient, zation immunoassays. These data demonstrate the 45 c) optionally initiating an examination of the patient prognostic utility in heart failure patients of measurement by a cardiologist, of very low circulating troponins at levels that are not d) recommending the initiation of the treatment or detectable with conventional assays, emphasizing the refraining from the treatment, optionally in consider- potential importance of the high sensitive troponin as- ation of the result of the patient’s examination by the says. 50 cardiologist. [0015] There is a need for further methods or means to identify risk patients before they receive treatment with [0020] Thus, the present invention provides the follow- anti-inflammatory drugs, particularly NSAIDs or steroids, ing items: more particularly selective Cox-2 inhibitors. Particularly, there is a need to provide a suitable diagnostic means. 55 (1) A method for diagnosing the risk of a patient to Particularly, there is a need for a diagnostic means that suffer from a cardiovascular complication as a con- allows to identify risk patients that have no history or no sequence of administration of a Cox-2 inhibiting known history of a cardiovascular risk or complication. compound, comprising the steps of

3 5 EP 2 133 696 A1 6

a) measuring the level of TROPONIN I OR T, f) markers of intravascular activation of coagu- b) diagnosing the risk of the patient by compar- lation ing the measured level to known levels associ- ated with different grades of risk in a patient. is measured. 5 (2) The method according to item (1), wherein the (12) The method according to any of items (1) to patient does not show obvious symptoms of a pre- (11), wherein the cardiovascular complication is cor- existing cardiovascular disease, risk or complication. onary heart disease, stable angina pectoris, acute coronary syndrome, unstable angina pectoris, myo- (3) The method according to item (1) or (2), wherein 10 cardial infarction, ST-elevated myocardial infarction, a plasma level of less than 3,7 pg/ml of TROPONIN non ST-elevated myocardial infarction, or stroke. I OR T is associated with no increased risk of suffer- ing from a cardiovascular complication. (13) The method according to any of items (1) to (12), wherein the level of TROPONIN I OR T is meas- (4) The method according to item (3), wherein the 15 ured in a urine, blood, blood plasma, or blood serum plasma level is less than 3,7 pg/ml. sample.

(5) The method according to item (1) or (2), wherein (14) The method according to any of items (1) to a plasma level of more than 3,7 and less than 10 (13), wherein the level of the TROPONIN I OR T is pg/ml of TROPONIN I OR T is associated with an 20 measured using a specifically binding ligand, an ar- increased risk of suffering from a cardiovascular ray, a microfluidic device, a chemiluminescence an- complication. alyzer, or a robotic device.

(6) The method according to item (1) or (2), wherein (15) The method according to item (13) or (14), a plasma level of more than 10 pg/ml of TROPONIN 25 wherein the specifically binding ligand is an antibody I OR T is associated with a highly increased risk of or an aptamer. suffering from a cardiovascular complication. (16) The method according to any of items (1) to (7) The method according to any of items (1) to (6), (15), wherein the cardiovascular risk in a patient who wherein the method is carried out within the moni- 30 is being treated with a selective Cox-2 inhibitor, is toring of a therapy with a selective Cox-2 inhibitor. monitored.

(8) The method according to item (7), wherein the (17) The method according to any of items (1) to method is for monitoring of an intermittent therapy (16), wherein the patient is a candidate for adminis- with a selective Cox-2 inhibitor. 35 tration of a selective Cox-2 inhibitor.

(9) The method according to item (8), wherein the (18) The method according to item (17), wherein the administration with the selective Cox-2 inhibitor is method is carried out before the selective Cox-2 in- stopped when the level of TROPONIN I OR T reach- hibitor is administered. es a prescribed value and is optionally re-initiated 40 when the level falls below the prescribed value. The (19) A method of deciding whether to initiate treat- prescribed value is suitably 3,7 pg/ml of TROPONIN ment of a patient with a compound having Cox-2 I OR T. inhibiting properties, which method comprises

(10) The method according to any of items (1) to (9), 45 a) measuring, in vitro, the level of TROPONIN I wherein the coxibe is chosen from the group con- OR T in the patient, sisting of , rofecoxib, , valdecox- b) diagnosing the risk of the patient by compar- ib, parecoxib, and lumiracoxib. ing the measured level with known level(s) as- sociated with different grades of risk in a patient (11) The method according to any of items (1) to 50 c) wherein, (10), wherein additionally the level(s) of at least one marker chosen from the group consisting of ca) if the risk is diagnosed not to be in- creased, then initiation of treatment is rec- a) markers of inflammation ommended, and/or b) markers of endothel function 55 cb) if the risk is diagnosed to be increased c) markers of ischemia or highly increased, then refraining from the d) markers of thrombocyte activation treatment is recommended, e) markers of atherosclerosis activation

4 7 EP 2 133 696 A1 8

optionally in consideration of the result of an ex- pound having Cox-2 inhibiting properties, to suffer from amination of the patient by a cardiologist. a cardiovascular complication. Preferably the level is de- termined in a body fluid or tissue sample of the patient. (20) The method according to item (19), wherein the [0025] The compound having Cox-2 inhibiting proper- compound is a selective Cox-2 inhibitor. 5 ties (also referred to as Cox-2 inhibitor) can also be any compound having anti-inflammatory properties (e.g. it (21) The method according to item (20), wherein the can be a steroid or NSAID). Most preferably, the com- selective Cox-2 inhibitor is chosen from the group pound is a selective Cox-2 inhibitor. consisting of celecoxib, rofecoxib, etoricoxib, val- [0026] The present invention provides simple and in- decoxib, parecoxib, and lumiracoxib. 10 expensive methods and means to screen patients who are about to receive with a compound having (22) The method according to any of items (19) to Cox-2 inhibiting properties, particularly a selective Cox- (21), wherein 2 inhibitor for their risk of developing a cardiovascular complication as a consequence of said medication. Said a) if the measured plasma or serum level of 15 medication may comprise any anti-inflammatory drug or TROPONIN I OR T is 3,7 pg/ml or higher, it is compound having Cox-2 inhibiting properties, in partic- recommended not to treat the patient or to ap- ular an NSAID, steroid, selective Cox-2 inhibitor, or any prove treatment under cardiovascular monitor- combination thereof. For example, said medication may ing and/or at low dosage of the selective Cox-2 comprise combined treatment with a steroid and a selec- inhibitor, and/or 20 tive Cox-2 inhibitor or it may comprise combined treat- b) if the measured measured plasma or serum ment with a selective Cox-2 inhibitor and another NSAID level of TROPONIN I OR T is 10 pg/ml or higher, (see also Example 5). The present invention also pro- then it is recommended not to treat the patient. vides levels of VERY LOW CONCENTRATIONS OF TROPONIN I OR T indicating the existence or severity (23) A method to determine whether treatment of a 25 of a cardiovascular risk in patients with or without obvious patient with an anti-inflammatory drug may be initi- symptoms of a cardiovascular disorder and/or a cardio- ated, said method comprising vascular disease. [0027] It may be assumed that patients with a cardio- a) measuring the level of TROPONIN I OR T in vascular disorder in their case history (myocardial infarc- a plasma or serum sample from the patient, 30 tion, unstable angina pectoris, coronary artery disease, b) wherein a measured plasma or serum level CABG (coronary artery bypass graft), stroke, pulmonary of TROPONIN I OR T of 3,7 pg/ml or higher, embolism, hypertrophy) or patients with hypertension or particularly of 10 pg/ml or higher, indicates that an underlying inflammatory disorder such as chronic pol- treatment should not be initiatied. yarthritis, osteoarthritis, or rheumatoid arthritis or other 35 rheumatoid disorders are predisposed to the occurrence (24) The method according to item (23) wherein the of cardiovascular complications. In rheumatoid arthritis, anti-inflammatory drug is a selective Cox-2 inhibitor. the cardiovascular death rate is particularly high for var- ious reasons. The term "stroke" relates to any cerebrov- [0021] The compound having Cox-2 inhibiting proper- ascular event in which blood flow to small or large regions ties (also referred to as Cox-2 inhibitor) can also be any 40 of the brain is interrupted, e.g due to haemorrhage into compound having anti-inflammatory properties (e.g. it the brain or a thrombosis of a cerebral artery. A stroke can be a steroid or NSAID). Most preferably, the com- may result in temporary loss of consciousness or paral- pound is a selective Cox-2 inhibitor. ysis. In this case, the stroke is termed "apoplectic stroke". [0022] From what was said earlier, it is evident that the [0028] Furthermore, it may be assumed that individu- methods are preferably carried out before the compound 45 als having a history of a cardiovascular disease (i.e. in- in question is administered. dividuals suffering from e.g.:coronary artery disease, [0023] The methods may be preceded by the step of ischemic heart disease, congestive heart failure, dilated taking a body fluid or tissue sample from the patient. The cardiomyopathy, stable angina pectoris (SAP) and indi- taking of the said body fluid or tissue sample can be car- viduals with acute coronary syndromes (ACS)) are pre- ried out by non-medical staff (i.e. not having an education 50 disposed to the occurrence of cardiovascular complica- necessary for carrying out the profession of a physician). tions. ACS patients can show unstable angina pectoris This applies in particular when the body sample is blood. (UAP) or these individuals have already suffered from a [0024] The object of the invention is also attained by myocardial infarction (MI). MI can be an ST-elevated MI use of a diagnostic means for measuring, preferably in or a non-ST-elevated MI. The occurring of an MI can be vitro, a patient’s level of VERY LOW CONCENTRA- 55 followed by a left ventricular dysfunction (LVD). Finally, TIONS OF TROPONIN I OR T, for diagnosing the cardi- LVD patients undergo congestive heart failure (CHF) with ovascular risk of a patient who is a candidate for admin- a mortality rate of roughly 15 %. istration, particularly future administration, of a com- [0029] Symptoms of cardiovascular complications/dis-

5 9 EP 2 133 696 A1 10 eases may include e.g. ECG signs like new Q-waves or arterial thrombosis or development of arterial thrombo- bundle branch block, signs of ischemic heart disease, sis. Arterial thrombosis may cause coronary syndromes acute coronary syndromes, acute myocardial infarction , (e.g. acute myocardial infarction or stroke). signs of non-fatal stroke, the onset or worsening of heart [0036] Particularly, "cardiovascular complication" re- failure as suggested by development of edema or wors- 5 lates to a complication in the arterial system, e.g. ACS, ening of preexistent edema of the lower extremities, rales UAP, MI, ST-elevated MI, non-ST-elevated MI, or stroke. on auscultation or pulmonary congestion, new onset of [0037] More particularly, "cardiovascular complica- arterial hypertension or worsening of preexistent arterial tion" relates to MI, ST-elevated MI, non-ST-elevated MI, hypertension, and venous thrombosis. or stroke. [0030] Symptoms of heart failure have been classified 10 [0038] Symptoms of cardiovascular diseases are into a functional classification system according to the known to the person skilled in the art and may include New York Heart Association (NYHA). Patients of Class e.g new Q-waves or bundle branch block, signs of non- I have no obvious symptoms of heart failure. Physical fatal stroke, the onset or worsening of heart failure as activity is not limited, and ordinary physical activity does suggested by development of edema or worsening of not cause undue fatigue, palpitation, or dyspnea (short- 15 preexistent edema of the lower extremities, rales on aus- ness of breath). Patients of class II have slight limitation cultation or pulmonary congestion, new onset of arterial of physical activity. They are comfortable at rest, but or- hypertension or worsening of preexistent arterial hyper- dinary physical activity results in fatigue, palpitation, or tension, and venous thrombosis. dyspnea. Patients of class III show a marked limitation [0039] Another characteristic of cardiovascular com- of physical activity. They are comfortable at rest, but less 20 plication or insufficiency can be the "left ventricular ejec- than ordinary activity causes fatigue, palpitation, or dys- tion fraction" (LVEF) which is also known as "ejection pnea. Patients of class IV are unable to carry out any fraction". People with a healthy heart usually have an physical activity without discomfort. They show symp- unimpaired LVEF, which is generally described as above toms of cardiac insufficiency at rest. If any physical ac- 50 %. Most people with a systolic heart disease which is tivity is undertaken, discomfort is increased. 25 symptomatic generally have an LVEF of 40 % or less. [0031] The present invention is particularly advanta- [0040] A cardiovascular complication (e.g. MI) may geous to identify apparently healthy patients showing no eventually result in an LVEF of 40% or less. symptoms of a preexisting cardiovascular disease or [0041] A cardiac insufficiency may either be "compen- complication but having an elevated risk to suffer from a sated" or "decompensated". Compensated means that cardiovascular complication. The present invention is al- 30 the regular oxygen need of the body can still be satisfied, so useful to confirm or monitor the risk status of the patient whereas decompensated means that the regular oxygen showing symptoms of a cardiovascular disease. need of the body is not satisfied anymore. [0032] The individuals may show clinical symptoms [0042] Patients with obvious or confirmed cardiovas- (e.g. dyspnea, chest pain, see also NYHA classification cular disease or disorder may per se be excluded from below) and they may be asymptomatic. Although the 35 the treatment with a compound having Cox-2 inhibiting present invention is particularly advantageous to identify activity, particularly a selective Cox2-inhibitor, in case apparently healthy patients showing no symptoms of a the patient shows symptoms which allow to stratify the preexisting cardiovascular disease or complication, but risk of the patient to suffer from a cardiovascular compli- having an elevated cardiovascular risk. The present in- cation, i.e. without measuring the level of VERY LOW vention is also useful in other settings, e.g to confirm or 40 CONCENTRATIONS OF TROPONIN I OR T. This ex- monitor the risk status of the patient showing symptoms clusion will in particular occur in patients suffering from of a cardiovascular disease. ischemic coronary heart diseases, having suffered from [0033] The term "cardiovascular risk" relates to the risk stroke, or patients classified according to NYHA class III- of developing a cardiovascular complication. IV. Even though a compound having Cox-2 inhibiting ac- [0034] The present invention relates to "cardiovascu- 45 tivity, particularly a selective Cox2-inhibitor, should not lar complications" developing as a consequence of ad- be administered to these patients, the measurement of ministration of an anti-inflammatory drug or Cox-2 inhib- VERY LOW CONCENTRATIONS OF TROPONIN I OR itor, particularly an NSAID, steroid, or selective Cox-2 T according to the invention may allow an individual risk inhibitor. Cardiovascular complications are also known stratification and initiation of a treatment of the patient as "cardiovascular events". In the following only or mostly 50 with a compound having Cox-2 inhibiting activity, partic- the term "cardiovascular complication" will be used. ularly a selective Cox2-inhibitor, and/or a supervision of [0035] The term "cardiovascular complication" is the treatment, if a need for the administration exists or is known to the person skilled in the art. Thus, a "cardio- highly recommended. vascular complication" according to the present invention [0043] In cases where the symptoms do not allow an relates to any kind of such cardiovascular complication 55 unambiguous classification of the patient (and, hence, of ischemic heart disease or heart failure (particularly not a decision to exclude the patient from the treatment acute heart failure) known to the person skilled in the art, with a compound having Cox-2 inhibiting activities), or in particularly the term refers to a complication related to cases where the patient does not show a recognisable

6 11 EP 2 133 696 A1 12 risk of suffering from a cardiovascular complication, an entially expressed (i.e. upregulated or downregulated) in individual risk assessment may be carried out after meas- presence or absence of a certain condition, disease, or uring VERY LOW CONCENTRATIONS OF TROPONIN complication. Usually, a molecular marker is defined as I OR T. The individual assessment is in particular carried a nucleic acid (such as an mRNA), whereas a biochem- out in patients belonging to risk groups, i.e. having a his- 5 ical marker is a protein or peptide. The level of a suitable tory of certain diseases or a life history which is known biochemical or molecular marker can indicate the pres- to augment the probability to suffer from a cardiovascular ence or absence of the condition, disease, risk, or com- complication. plication, and thus allow diagnosis. [0044] The individual risk assessment is in particular [0048] The present invention particularly takes advan- beneficial in the following patient groups: patients clas- 10 tage of VERY LOW CONCENTRATIONS OF TROPON- sified according to NYHA class I-II, patients suffering IN I OR T as a biochemical marker. It should be noted from diabetes, hyperlipidemia, hypertonie, in smokers that VERY LOW CONCENTRATIONS OF TROPONIN I and elderly patients, e.g. those having an age of 55 years OR T may be secreted in response to ischemia. or higher. [0049] VERY LOW CONCENTRATIONS OF TRO- [0045] The use of VERY LOW CONCENTRATIONS 15 PONIN I OR T is induced and secreted from the heart OF TROPONIN I OR T as molecular or biochemical mark- following acute and chronic stimulation associated with ers is known as such. In WO 02/089657, it has been ischemia, hypertension, pressure overload, and cardiac suggested to measure brain natriuretic peptide (BNP) to dysfunction. diagnose various myocardial dysfunctions. In WO [0050] Determining the amount of VERY LOW CON- 02/083913 it has been suggested to use BNP to predict 20 CENTRATIONS OF TROPONIN I OR T or any other pep- near-term morbidity or mortality in patients with non-ST- tide or polypeptide referred to in this specification relates elevated acute coronary syndromes. In a previously un- to measuring the amount or concentration, preferably published European application (EP 1 577 673 A1) it has semi-quantitatively or quantitatively. Measuring can be been suggested to use natriuretic peptides to diagnose done directly or indirectly. Direct measuring relates to the risk of a patient of suffering from a cardiovascular 25 measuring the amount or concentration of the peptide or complication as a consequence of an increase of intra- polypeptide based on a signal which is obtained from the vasal volume. peptide or polypeptide itself and the intensity of which [0046] The present invention is particularly advanta- directly correlates with the number of molecules of the geous to general practitioners, specialized physicians, peptide present in the sample. Such a signal - sometimes and specialized wards, departments, or clinics which fre- 30 referred to herein as intensity signal -may be obtained, quently have no access to extensive cardiological exam- e.g., by measuring an intensity value of a specific physical ination by cardiologists. The present invention provides or chemical property of the peptide or polypeptide. Indi- means and methods to such non-cardiologists for simple rect measuring includes measuring of a signal obtained and reliable screening of patients and identification of from a secondary component (i.e. a component not being those patients who are posed at a cardiovascular risk 35 the peptide or polypeptide itself) or a biological read out with respect to administration of a Cox-inhibitor or an system, e.g., measurable cellular responses, ligands, la- anti-inflammatory drug, in particular with respect to ad- bels, or enzymatic reaction products. ministration of an NSAID, steroid, or a selective Cox-2 [0051] In accordance with the present invention, de- inhibitor. Preferably, the decision about continuing or in- termining the amount of a peptide or polypeptide can be itiating treatment with the Cox-2 inhibitor, in particular the 40 achieved by all known means for determining the amount NSAID, steroid, or selective Cox-2 inhibitor, will be made of a peptide in a sample. Said means comprise immu- by a physician. More preferably, the decision about con- noassay devices and methods which may utilize labeled tinuing or initiating treatment with the Cox-2 inhibitor, in molecules in various sandwich, competition, or other as- particular the NSAID, steroid, or selective Cox-2 inhibitor, say formats. Said assays will develop a signal which is will be made by a cardiologist or after consulting a car- 45 indicative for the presence or absence of the peptide or diologist. This in particular applies in cases where high polypeptide. Moreover, the signal strength can, prefera- levels of VERY LOW CONCENTRATIONS OF TRO- bly, be correlated directly or indirectly (e.g. reverse- pro- PONIN I OR T are measured. In cases in which the patient portional) to the amount of polypeptide present in a sam- is diagnosed to have no cardiovascular risk, a decision ple. Further suitable methods comprise measuring a to continue or initiate treatment with a Cox-2 inhibiting 50 physical or chemical property specific for the peptide or compound, particularly an NSAID, steroid, or selective polypeptide such as its precise molecular mass or NMR Cox-2 inhibitor, may be made by a physician other than spectrum. Said methods comprise, preferably, biosen- a cardiologist. sors, optical devices coupled to immunoassays, bio- [0047] The invention takes advantage of certain bio- chips, analytical devices such as mass- spectrometers, chemical or molecular markers. The terms "biochemical 55 NMR- analyzers, or chromatography devices. Further, marker" and "molecular marker" are known to the person methods include micro-plate ELISA-based methods, ful- skilled in the art. In particular, biochemical or molecular ly-automated or robotic immunoassays (available for ex- markers are gene expression products which are differ- ample on Elecsys™ analyzers), CBA (an enzymatic Co-

7 13 EP 2 133 696 A1 14 balt Binding Assay, available for example on Roche-Hi- sequences of a human acceptor antibody. The donor se- tachi™ analyzers), and latex agglutination assays (avail- quences will usually include at least the antigen-binding able for example on Roche-Hitachi™ analyzers). amino acid residues of the donor but may comprise other [0052] Preferably, determining the amount of a peptide structurally and/or functionally relevant amino acid resi- or polypeptide comprises the steps of (a) contacting a 5 dues of the donor antibody as well. Such hybrids can be cell capable of eliciting a cellular response the intensity prepared by several methods well known in the art. Pref- of which is indicative of the amount of the peptide or erably, the ligand or agent binds specifically to the peptide polypeptide with the said peptide or polypeptide for an or polypeptide. Specific binding according to the present adequate period of time, (b) measuring the cellular re- invention means that the ligand or agent should not bind sponse. For measuring cellular responses, the sample 10 substantially to ("cross-react" with) another peptide, or processed sample is, preferably, added to a cell culture polypeptide or substance present in the sample to be and an internal or external cellular response is measured. analyzed. Preferably, the specifically bound peptide or The cellular response may include the measurable ex- polypeptide should be bound with at least 3 times higher, pression of a reporter gene or the secretion of a sub- more preferably at least 10 times higher and even more stance, e.g. a peptide, polypeptide, or a small molecule. 15 preferably at least 50 times higher affinity than any other The expression or substance shall generate an intensity relevant peptide or polypeptide. Nonspecific binding may signal which correlates to the amount of the peptide or be tolerable, if it can still be distinguished and measured polypeptide. unequivocally, e.g. according to its size on a Western [0053] Also preferably, determining the amount of a Blot, or by its relatively higher abundance in the sample. peptide or polypeptide comprises the step of measuring 20 Binding of the ligand can be measured by any method a specific intensity signal obtainable from the peptide or known in the art. Preferably, said method is semi-quan- polypeptide in the sample. As described above, such a titative or quantitative. Suitable methods are described signal may be the signal intensity observed at an m/z in the following. variable specific for the peptide or polypeptide observed [0055] First, binding of a ligand may be measured di- in mass spectra or a NMR spectrum specific for the pep- 25 rectly, e.g. by NMR or surface plasmon resonance. tide or polypeptide. [0056] Second, if the ligand also serves as a substrate [0054] Determining the amount of a peptide or of an enzymatic activity of the peptide or polypeptide of polypeptide may, preferably, comprise the steps of (a) interest, an enzymatic reaction product may be meas- contacting the peptide with a specific ligand, (b) (option- ured (e.g. the amount of a protease can be measured by ally) removing non-bound ligand, (c) measuring the 30 measuring the amount of cleaved substrate, e.g. on a amount of bound ligand. The bound ligand will generate Western Blot). Alternatively, the ligand may exhibit en- an intensity signal. Binding according to the present in- zymatic properties itself and the "ligand/peptide or vention includes both covalent and non-covalent binding. polypeptide" complex or the ligand which was bound by A ligand according to the present invention can be any the peptide or polypeptide, respectively, may be contact- compound, e.g., a peptide, polypeptide, nucleic acid, or 35 ed with a suitable substrate allowing detection by the small molecule, binding to the peptide or polypeptide de- generation of an intensity signal. For measurement of scribed herein. Preferred ligands include antibodies, nu- enzymatic reaction products, preferably the amount of cleic acids, peptides or polypeptides such as receptors substrate is saturating. The substrate may also be la- or binding partners for the peptide or polypeptide and beled with a detectable lable prior to the reaction. Pref- fragments thereof comprising the binding domains for 40 erably, the sample is contacted with the substrate for an the peptides, and aptamers, e.g. nucleic acid or peptide adequate period of time. An adequate period of time re- aptamers. Methods to prepare such ligands are well- fers to the time necessary for a detectable, preferably known in the art. For example, identification and produc- measurable, amount of product to be produced. Instead tion of suitable antibodies or aptamers is also offered by of measuring the amount of product, the time necessary commercial suppliers. The person skilled in the art is fa- 45 for appearance of a given (e.g. detectable) amount of miliar with methods to develop derivatives of such ligands product can be measured. with higher affinity or specificity. For example, random [0057] Third, the ligand may be coupled covalently or mutations can be introduced into the nucleic acids, pep- non-covalently to a label allowing detection and meas- tides or polypeptides. These derivatives can then be test- urement of the ligand. Labeling may be done by direct or ed for binding according to screening procedures known 50 indirect methods. Direct labeling involves coupling of the in the art, e.g. phage display. Antibodies as referred to label directly (covalently or non-covalently) to the ligand. herein include both polyclonal and monoclonal antibod- Indirect labeling involves binding (covalently or non-cov- ies, as well as fragments thereof, such as Fv, Fab and F alently) of a secondary ligand to the first ligand. The sec- (ab)2 fragments that are capable of binding antigen or ondary ligand should specifically bind to the first ligand. hapten. The present invention also includes single chain 55 Said secondary ligand may be coupled with a suitable antibodies and humanized hybrid antibodies wherein label and/or be the target (receptor) of tertiary ligand bind- amino acid sequences of a non-human donor antibody ing to the secondary ligand. The use of secondary, terti- exhibiting a desired antigen-specificity are combined with ary or even higher order ligands is often used to increase

8 15 EP 2 133 696 A1 16 the signal. Suitable secondary and higher order ligands a solid support comprising a ligand for the peptide or may include antibodies, secondary antibodies, and the polypeptide as specified above with a sample comprising well-known streptavidin-biotin system (Vector Laborato- the peptide or polypeptide and (b) measuring the amount ries, Inc.). The ligand or substrate may also be "tagged" peptide or polypeptide which is bound to the support. The with one or more tags as known in the art. Such tags may 5 ligand, preferably chosen from the group consisting of then be targets for higher order ligands. Suitable tags nucleic acids, peptides, polypeptides, antibodies and include biotin, digoxygenin, His-Tag, Glutathion-S- aptamers, is preferably present on a solid support in im- Transferase, FLAG, GFP, myc-tag, influenza A virus hae- mobilized form. Materials for manufacturing solid sup- magglutinin (HA), maltose binding protein, and the like. ports are well known in the art and include, inter alia, In the case of a peptide or polypeptide, the tag is prefer- 10 commercially available column materials, polystyrene ably at the N-terminus and/or C-terminus. Suitable labels beads, latex beads, magnetic beads, colloid metal parti- are any labels detectable by an appropriate detection cles, glass and/or silicon chips and surfaces, nitrocellu- method. Typical labels include gold particles, latex lose strips, membranes, sheets, duracytes, wells and beads, acridan ester, luminol, ruthenium, enzymatically walls of reaction trays, plastic tubes etc. The ligand or active labels, radioactive labels, magnetic labels ("e.g. 15 agent may be bound to many different carriers. Examples magnetic beads", including paramagnetic and superpar- of well-known carriers include glass, polystyrene, poly- amagnetic labels), and fluorescent labels. Enzymatically vinyl chloride, polypropylene, polyethylene, polycar- active labels include e.g. horseradish peroxidase, alka- bonate, dextran, nylon, amyloses, natural and modified line phosphatase, beta-Galactosidase, Luciferase, and celluloses, polyacrylamides, agaroses, and magnetite. derivatives thereof. Suitable substrates for detection in- 20 The nature of the carrier can be either soluble or insoluble clude di-amino-benzidine (DAB), 3,3’-5,5’-tetramethyl- for the purposes of the invention. Suitable methods for benzidine, NBT-BCIP (4-nitro blue tetrazolium chloride fixing/immobilizing said ligand are well known and in- and 5-bromo-4-chloro-3-indolyl-phosphate, available as clude, but are not limited to ionic, hydrophobic, covalent ready-made stock solution from Roche Diagnostics), interactions and the like. It is also contemplated to use CDP-Star™ (Amersham Biosciences), ECF™ (Amer- 25 "suspension arrays" as arrays according to the present sham Biosciences). A suitable -substrate com- invention (Nolan 2002, Trends Biotechnol. 20(1):9-12). bination may result in a colored reaction product, fluo- In such suspension arrays, the carrier, e.g. a microbead rescence or chemoluminescence, which can be meas- or microsphere, is present in suspension. The array con- ured according to methods known in the art (e.g. using sists of different microbeads or microspheres, possibly a light-sensitive film or a suitable camera system). As for 30 labeled, carrying different ligands. Methods of producing measuring the enyzmatic reaction, the criteria given such arrays, for example based on solid-phase chemistry above apply analogously. Typical fluorescent labels in- and photo-labile protective groups, are generally known clude fluorescent proteins (such as GFP and its deriva- (US 5,744,305). tives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa [0059] The term "amount" as used herein encompass- dyes (e.g. Alexa 568). Further fluorescent labels are 35 es the absolute amount of a polypeptide or peptide, the available e.g. from Molecular Probes (Oregon). Also the relative amount or concentration of the said polypeptide use of quantum dots as fluorescent labels is contemplat- or peptide as well as any value or parameter which cor- ed. Typical radioactive labels include 35S, 125I, 32P, 33P relates thereto or can be derived therefrom. Such values and the like. A radioactive label can be detected by any or parameters comprise intensity signal values from all method known and appropriate, e.g. a light-sensitive film 40 specific physical or chemical properties obtained from or a phosphor imager. Suitable measurement methods the said peptides by direct measurements, e.g., intensity according the present invention also include precipitation values in mass spectra or NMR spectra. Moreover, en- (particularly immunoprecipitation), electrochemilumi- compassed are all values or parameters which are ob- nescence (electro-generated chemiluminescence), RIA tained by indirect measurements specified elsewhere in (radioimmunoassay), ELISA (enzyme-linked immuno- 45 this description, e.g., response levels determined from sorbent assay), sandwich enzyme immune tests, elec- biological read out systems in response to the peptides trochemiluminescence sandwich immunoassays or intensity signals obtained from specifically bound lig- (ECLIA), dissociation-enhanced lanthanide fluoro immu- ands. It is to be understood that values correlating to the no assay (DELFIA), scintillation proximity assay (SPA), aforementioned amounts or parameters can also be ob- turbidimetry, nephelometry, latex-enhanced turbidimetry 50 tained by all standard mathematical operations. or nephelometry, or solid phase immune tests. Further [0060] The term "comparing" as used herein encom- methods known in the art (such as gel electrophoresis, passes comparing the amount of the peptide or polypep- 2D gel electrophoresis, SDS polyacrylamid gel electro- tide comprised by the sample to be analyzed with an phoresis (SDS-PAGE), Western Blotting, and mass amount of a suitable reference source specified else- spectrometry), can be used alone or in combination with 55 where in this description. It is to be understood that com- labeling or other dectection methods as described above. paring as used herein refers to a comparison of corre- [0058] The amount of a peptide or polypeptide may sponding parameters or values, e.g., an absolute amount be, also preferably, determined as follows: (a) contacting is compared to an absolute reference amount while a

9 17 EP 2 133 696 A1 18 concentration is compared to a reference concentration od of the present invention. or an intensity signal obtained from a test sample is com- [0062] A preferred threshold (i.e. reference amount) pared to the same type of intensity signal of a reference for VERY LOW CONCENTRATIONS OF TROPONIN I sample. The comparison referred to in step (b) of the OR T is at least one to two times the ULN. The ULN method of the present invention may be carried out man- 5 referred to in this context is, preferably, 1 pg/ml. ually or computer assisted. For a computer assisted com- [0063] Thus, the reference amount defining a thresh- parison, the value of the determined amount may be com- old amount for VERY LOW CONCENTRATIONS OF pared to values corresponding to suitable references TROPONIN I OR T as referred to in accordance with the which are stored in a database by a computer program. present invention is < 2 pg/ml, preferably 3,7 pg/ml. The computer program may further evaluate the result 10 [0064] An amount of VERY LOW CONCENTRA- of the comparison, i.e. automatically provide the desired TIONS OF TROPONIN I OR T larger than the reference assessment in a suitable output format. Based on the amount is, more preferably, indicative for a subject which comparison of the amount determined in step a) and the should be admitted to hospital. reference amount, it is possible to assess whether a sub- [0065] The term "variant" also relates to a post-trans- ject is susceptible for a cardiac intervention, i.e. belong- 15 lationally modified peptide such as glycosylated peptide. ing to the group of subjects which can be successfully A "variant" is also a peptide which has been modified treated by the cardiac intervention. Therefore, the refer- after collection of the sample, for example by covalent or ence amount is to be chosen so that either a difference non-covalent attachment of a label, particularly a radio- or a similarity in the compared amounts allows identifying active or fluorescent label, to the peptide. those the test subject which belong into the group of sub- 20 [0066] The term "diagnosing" is known to the person jects susceptible for cardiac intervention or identifying skilled in the art. Diagnosing is understood as becoming those test subjects which are not susceptible for a cardiac aware of a particular medical condition, disease, compli- intervention. cation, or risk. Diagnosing may also be understood as [0061] Accordingly, the term "reference amount" as detecting or determining the presence of a particular used herein refers to an amount which allows assessing 25 medical condition, disease, complication, or risk. Diag- whether a subject is to be admitted to the hospital or can nosing according to the present invention also includes be discharged to home. Accordingly, the reference may monitoring, confirmation, subclassification and predic- e.g. be derived from (i) a subject known to have been tion of the relevant disease, complication, or risk. Moni- successfully admitted to the hospital, i.e. who has been toring relates to keeping track of an already diagnosed subject to further examination and subsequent or inten- 30 risk or complication, e.g. to analyze an elevation or de- sive care treatment based on the results of the further crease of the risk or the influence of a particular treatment investigation without the occurrence of adverse effects on the elevation or decrease of the risk. Confirmation such as mortality or side effects caused by unadapted relates to the strengthening or substantiating a diagnosis treatment regimen, or (ii) a subject known to have not already performed using other indicators or markers. been admitted to the hospital and which died or devel- 35 Subclassification relates to further defining a diagnosis oped side effects caused by unadapted treatment regi- according to different subclasses of the diagnosed dis- men. Moreover, the reference amount may define a ease risk, e.g. defining according to increased risk or threshold amount, whereby an amount larger than the highly increased risk. Subclassification is also referred threshold shall be indicative for a subject which should to as "risk stratification". Prediction relates to assessing be admitted to the hospital for further examination and/or 40 a cardiovascular risk before other symptoms or markers (intensive) treatment, while an amount lower than the have become evident or have become significantly al- threshold amount shall be an indicator for a subject which tered. can be discharged to home. The reference amount ap- [0067] "Diagnosing the cardiovascular risk" according plicable for an individual subject may vary depending on to the present invention includes diagnosing (i.e. becom- various physiological parameters such as age, gender, 45 ing aware of) a cardiovascular risk, but also monitoring or subpopulation, as well as on the means used for the an elevation or decrease of a pre-existing or known car- determination of the polypeptide or peptide referred to diovascular risk ("risk stratification"). herein. A suitable reference amount may be determined [0068] The term "patient" according to the present in- from a reference sample to be analyzed together, i.e. vention relates to a healthy individual, an apparently simultaneously or subsequently, with the test sample. A 50 healthy individual, or particularly an individual suffering preferred reference amount serving as a threshold may from a disease. The patient can be both a male or a fe- be derived from the upper limit of normal (ULN), i.e. the male individual, as the present invention is suited for di- upper limit of the physiological amount to be found in a agnosing both groups. Particularly, the patient is suffer- population of apparently healthy subjects. The ULN for ing from and/or treated for rheumatism, rheumatoid ar- a given population of subjects can be determined by var- 55 thritis (chronic polyarthritis), osteoarthritis, psoriatic ar- ious well known techniques. A suitable technique may thritis, spondyloarthropathy, or other inflammatory dis- be to determine the median of the population for the pep- eases or osteoarthrosis. The patient may also be suffer- tide or polypeptide amounts to be determined in the meth- ing from ulcerations or cancer. In this context it should

10 19 EP 2 133 696 A1 20 be noticed that the aforementioned diseases are diseas- In a particular example, the level of VERY LOW CON- es in which administration of compounds having Cox-2 CENTRATIONS OF TROPONIN I OR T may be moni- inhibiting properties (as anti-inflammatory drugs), in par- tored before and during administration of the anti-inflam- ticular NSAIDs, steroids, or selective Cox-2 inhibitors is matory drug. If the level of VERY LOW CONCENTRA- considered as part of the treatment. Even more particu- 5 TIONS OF TROPONIN I OR T increases upon beginning larly, the patient has no known history of cardiovascular of treatment and decreases upon discontinuing treat- risk, complication or disease, and/or no or little symptoms ment with the drug, it indicates that the drug is causing of a cardiovascular risk, complication or disease, and/or an elevation of the cardiovascular risk. Similarly, if the he is not being treated for a cardiovascular disease, risk, level correlates with the dosage of the drug, it also indi- or complication. However, also healthy individuals who 10 cates that the drug is causing an elevation of the cardi- have no signs or history of a cardiovascular risk, compli- ovascular risk. Any cardiovascular complication mani- cation or disease are considered to be patients according festing under such circumstances is likely to be caused to the present invention. by the administration of the drug. This is even more likely, [0069] Preferably, the patient is treated or is about to if the increase of the level of the VERY LOW CONCEN- be treated with an anti-inflammatory drug. Such a patient 15 TRATIONS OF TROPONIN I OR T upon administration is understood to be a "candidate" for treatment with said of the drug is unusually high compared to other patients drug. More preferably, the patient is treated or is about or if a small increase in dosage causes an unusually to be treated with a selective Cox-2 inhibitor. Such a pa- steep increase in the level of the VERY LOW CONCEN- tient is understood to be a "candidate" for treatment with TRATIONS OF TROPONIN I OR T. a selective Cox-2 inhibitor. Of course, treatment with the 20 [0072] Anti-inflammatory drugs are known to the per- respective drug or selective Cox-2 inhibitor should in gen- son skilled in the art. Particularly, such drugs include non- eral be considered an advisable treatment option. steroid anti-rheumatics (also known as non-steroidal an- [0070] It is known to the person skilled in the art, under ti-inflammatory drugs, NSAIDs), Cox-2 inhibitors, corti- what circumstances a cardiovascular complication can costeroids, and TNF inhibitors. be considered to occur "as a consequence" of adminis- 25 [0073] Examples for anti-inflammatory drugs include tration of an anti-inflammatory drug or a Cox-2 inhibitor, ; Alclometasone Dipropionate; Algestone Ac- in particular of an NSAID, steroid, or selective Cox-2 in- etonide; Alpha Amylase; Amcinafal; Amcinafide; Am- hibitor. It should be understood that it may not be possible fenac Sodium; Amiprilose Hydrochloride; Anakinra; An- or necessary to definitively establish the causal relation- irolac; ; Apazone; Balsalazide Disodium; ship. It may be sufficient to establish a sufficiently high 30 ; ; Hydrochloride; probability that the cardiovascular complication was or Bromelains; Broperamole; Budesonide; ; will be precipitated by administration of the anti-inflam- Cicloprofen; Cintazone; Cliprofen; Clobetasol Propion- matory drug or Cox-2 inhibitor, in particular an NSAID, ate; Clobetasone Butyrate; Clopirac; Cloticasone Propi- steroid, or selective Cox-2 inhibitor. E.g., the APPROVE onate; Cormethasone Acetate; Cortodoxone; Celecoxib; study indicates an almost fourfold increase in the number 35 Rofecoxib (VIOXX); Etoricoxib; Valdecoxib; Parecoxib; of cardiovascular complications between the test and the lumiracoxib; Deflazacort; Desonide; Desoximetasone; placebo group. For the purpose of the present invention, Dexamethasone Dipropionate; ; Diclofenac it may be justified to assume that any cardiovascular com- Potassium; Diclofenac Sodium; Diflorasone Diacetate; plication in a patient being treated with a selective Cox- Diflumidone Sodium; ; Difluprednate; Diftalone; 2 inhibitor is also caused by the treatment. Thus, the 40 ; Drocinonide; Endrysone; Enlimom- present invention also relates to diagnosing the risk to ab; Enolicam Sodium; ; ; ; suffer from a cardiovascular complication after adminis- ; Fenamole; ; ; Fenclorac; tration of an anti-inflammatory drug, Cox-2 inhibitor, Fendosal; Fenpipalone; ; Flazalone; Fluaza- NSAID, or selective Cox-2 inhibitor. cort; ; ; Flunisolide Acetate; [0071] Furthermore, the person skilled in the art is fa- 45 ; Flunixin Meglumine; Fluocortin Butyl; miliar with methods or signs indicating a causal relation- Fluormetholone Acetate; Fluquazone; ; ship. For example, a time relationship between adminis- Fluretofen; Fluticasone Propionate; Furaprofen; Furo- tration of the drug and the manifestation of a cardiovas- bufen; Halcinonide; Halobetasol Propionate; Halopre- cular complication is always an indicator that the drug done Acetate; Ibufenac; ; Ibuprofen Aluminium; has been causal for the complication or "precipitated" the 50 Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin complication. E.g. the increase in the number of cardio- Sodium; ; Indoxole; Intrazole; Isoflupredone vascular complications in the APPROVE study became Acetate; Isoxepac; ; ; Lofemizole Hy- evident after 18 months of treatment. In another example, drochloride; ; Loteprednol Etabonate; if the severity of the cardiovascular complication corre- Meclofenamate Sodium; ; Meclori- lates with the amount of the drug administered, it may 55 sone Dibutyrate; ; (Mobic™); indicate a causal relationship. For example, pathological Mesalamine; Meseclazone; Methylprednsisolone Sulep- changes in an echocardiogram or electrocardiogram tanate; Momiflumate; ; Naproxen; Naprox- may improve upon discontinuing treatment with the drug. en Sodium; Naproxol; Nimazone; Olsalazine Sodium;

11 21 EP 2 133 696 A1 22

Orgotein; Orpanoxin; ; ; marker for the enzymatic function of Cox-1 is the forma- Paranyline Hydrochloride; Pentosan Polysulfate Sodi- tion of thromboxan A2, whereas a typical marker for the um; Phenbutazone Sodium Glycerate; Pirfenidone; enzymatic function of Cox-2 is the formation of prostag- ; Piroxicam Cinnamate; Piroxicam Olamine; landins (e.g. E2 from macrophages. ; Prednazate; Prifelone; Prodolic Acid; Proqua- 5 [0079] Examples for a suitable test systems have been zone; Proxazole; Proxazole Citrate; Rimexolone; Roma- published (e.g. Warner, T.D., Giuliano, F., Vojnovic, I., zarit; Salcolex; Salnacedin; ; Salycilates; San- et al. (1999). Nonsteroid drug selectivities for cyclo-oxy- guinarium Chloride; Seclazone; Sermetacin; Sudoxi- genase-1 rather than cyclo-oxygenase-2 are associated cam; ; ; Talmetacin; Talniflumate; Ta- with human gastrointestinal toxicity: A full in vitro analy- losalate; Tebufelone; ; Tenidap Soidum; Tenox- 10 sis. Proceedings of the National Academy of Sciences icam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tix- USA, vol. 96., pp. 7563-7568, a relevant erratum has ocortol Pivalate; ; Tolmetin Sodium; Triclonide; been published in vol. 96(17), p. 9966d). This assay will Triflumidate; Zidometacin; Sodium; Etaner- be referred to as the William Harvey Modified Assay. The cept; Lenercept; Infliximab; Cortisone; Fluocortolone; assay is described in detail in Warner T.D., et al. supra, Hydrocortisone; Methyl-prednisolone; Prednisolone; 15 on page 7563-4, the description of which is expressly Prednisone; Prednylidene. incorporated herein by reference. [0074] As the case may be, it is comprised in the [0080] Preferably, a selective Cox-2 inhibitor accord- present invention to measure VERY LOW CONCEN- ing to the present invention is more than 5-fold Cox-2 TRATIONS OF TROPONIN I OR T also before adminis- selective according to the William Harvey Modified As- tering any of the above-mentioned compounds. 20 say, more preferably more than 50-fold Cox-2 selective [0075] The term "non-steroidal anti-rheumatics" (also according to the William Harvey Modified Assay (see referred to as non-steroidal anti-inflammatory drugs or Warner, T.D. et al., supra, Fig. 3 on page 7567). NSAIDs) is known to the person skilled in the art. NSAIDs [0081] Alternatively, the selective Cox-2 inhibitor ac- inhibit (also known as prostaglandin- cording to the present invention is a compound preferably H-synthetases). Cyclooxygenases catalyze the reaction 25 being more selective for Cox-2 than diclofenac, more from to (a cyclic en- preferably being more selective for Cox-2 than doperoxide), which is the precursor of prostaglandin I2 , even more preferably at least as selective (also known as prostacycline), thromboxan A2, and other as for Cox-2 as celecoxib under therapeutic conditions. . Prostaglandins play a significant role in [0082] In another preferred embodiment, the present pain, fever, and inflammatory reactions. There are two 30 invention relates to means and methods for diagnosing isoforms of cyclooxygenases, Cox-1 and Cox-2. The the cardiovascular risk of a patient who is a candidate Cox-2 gene is an immediate early gene and is induced for administration of a "coxibe". Examples for coxibes under conditions of tissue damage, pain reactions, or in- include celecoxib (Celebrex™, Pfizer), rofecoxib (VI- flammatory reactions. Thus, NSAIDs include Cox-1 in- OXX™, Merck), etoricoxib, valdecoxib, parecoxib (a pro- hibitors and Cox-2 inhibitors. The NSAIDs may inhibit 35 drug of valdecoxib), lumiracoxib (Prexige™, Novartis). both isoforms or they may be selective for one isoform Other similar compounds, several of which are under de- (i.e. they inhibit only one of the two isoforms at the ther- velopment and examination, are also included in the apeutic dosage). scope of the present invention. [0076] Examples for unspecific NSAIDs include Ibu- [0083] The currently discussed pathomechanism for profen; Flurbiprofen; Naproxen; Flufenamic Acid; 40 the undesired cardiovascular side-effects of selective Mefenamic Acid; Piroxicam; Diclofenac; Phenbutazone Cox-2 inhibitors may be as follows: The inhibition of pros- Sodium Glycerate; ; . taglandin I2 (PGI2) formation is attributed a major role in [0077] Selective Cox-2 inhibitors according to the the genesis of these cardiovascular incidents. PGI2 leads present invention are compounds which, under thera- to inhibition of thrombocyte aggregation, vasodilatation peutic conditions, do inhibit expression or, preferably, the 45 and prevention of the proliferation of smooth muscle cells enzymatic function of Cox-2, whereas not significantly in vitro. inhibiting expression or, preferably, the enzymatic func- [0084] On the other hand, A2, the most tion of Coy-1. Examples for selective Cox-2 inhibitors in- important COX-1 product of platelets leads to platelet clude coxibes (e.g. celecoxib, rofecoxib, etoricoxib, val- aggregation, vasoconstriction and vascular proliferation. decoxib, parecoxib (a pro-drug of valdecoxib), lumiracox- 50 It is suspected that the interference with the balanced ib), meclofenatmate, sulindac sulphide, diclofenac, equilibrium between PGI2 and promotes nimesulide, meloxicam, etodolac, NS398, L-745,337, intravascular activation of hemostasis, leads to an in- DFP (3-(2-propyloxy)-4-(4-methylsulphonylphenyl)-5,5- crease in blood pressure and enhances the acceleration dimethylfuranone). The latter three compounds are de- of atherosclerosis. scribed in Warner, T.D., et al., 1999. 55 [0085] Thus, in a preferred embodiment, the present [0078] The enzymatic function of the two cyclooxyge- invention also relates to the use of VERY LOW CON- nases can be measured according to methods known in CENTRATIONS OF TROPONIN I OR T for assessing the art, including suitable in vivo or in vitro tests. A typical the cardiovascular risk of a patient with respect to the

12 23 EP 2 133 696 A1 24 administration of an anti-inflammatory drug wherein the ity C-reactive protein). cardiovascular risk is caused by a disturbance of the bal- [0091] Markers of endothel function according to the anced equilibrium between Cox-1 and Cox-2 inhibition, present invention comprise any markers indicative of in- and/or the prostaglandin metabolism and/or the bal- travascular repair processes, including cytokines related 5 anced equilibrium between PGI2 and thromboxane A2, to such processes. Examples for such markers include more particularly the inhibition of the PGI2 formation. The selectins (e.g. E-selectin, P-selectin), ICAM-1 (intercel- present invention also relates to the use of VERY LOW lular cell adhesion molecule-1), VCAM-1 (vascular cell CONCENTRATIONS OF TROPONIN I OR T for assess- adhesion molecule-1), PDGF (platelet-derived growth ing the cardiovascular risk of a patient with respect to the factor), TGF-alpha, TGF-beta, catecholamines, prostag- administration of an anti-inflammatory drug which pro- 10 landins, angiotensin, endothelin, NO (nitric oxide). Al- motes intravascular thrombosis, and/or leads to an in- though already mentioned as being markers of inflam- crease in blood pressure and/or enhances the acceler- mation, TNF-alpha, TNF-beta, and IL-1 may also be con- ation of atherosclerosis. In a further embodiment, the sidered to belong to the group of markers of endothel present invention does not relate to the use of VERY function. LOW CONCENTRATIONS OF TROPONIN I OR T for 15 [0092] Markers of ischemia according to the present assessing the risk of suffering from a cardiovascular com- invention comprise any markers indicative of insufficient plication wherein the risk is due to an increase of blood oxygen supply resulting in ischemia. Particularly, the in- volume or intravasal volume (volume overload). sufficient oxygen supply is local and any resulting [0086] In the present context, the term "steroid" is used ischemia is also local. Such local ischemia may be as an abbreviation of the term "corticosteroid". It is gen- 20 caused by arterial thrombosis limiting the blood flow to erally thought that corticosteroids exert their anti-inflam- the target tissue of the affected artery. Examples for matory activity by influencing the prostaglandin metabo- markers of ischemia include ischemic modified albumin lism. Examples for corticosteroids according to the (IMA) and high-sensitive troponins (e.g. TnI and TnT). present invention include cortisone; fluocortolone; hydro- [0093] Markers of thrombocyte activation according to cortisone; methyl-prednisolone; prednisolone; pred- 25 the present invention comprise any markers indicative of nisone; prednylidene. activation or aggregability of blood platelets. A preferred [0087] TNF inhibitors are also used as anti-inflamma- marker is soluble CD40 ligand (sCD40L). It should be tory drugs, in particular in patients with rheumatoid ar- understood that thrombocyte activation or platelet aggre- thritis (increased concentrations of TNF-α were found in gability may also be measured by analysis of a thrombo- patients suffering from rheumatoid arthritis). Inhibition of 30 cyte-rich blood plasma sample. After taking the sample, TNF-α also reduces the formation of IL-1 and IL-6. Ex- a trigger substance is added and the time-course of ag- amples for TNF inhibitors include Etanercept, Lenercept gregation of thrombocytes is measured by the reduction (an analogon of Etanercept), Infliximab, and D2E7 (a in turbidity. The reduction in turbidity is caused by the completely humanized monoclonal antibody against aggregation of the many small platelets into larger ag- TNF-α). 35 gregates, which reduces the light scatter. The trigger sub- [0088] In another preferred embodiment, the present stance may be any substance deemed appropriate by invention relates to additionally measuring the level of at the person skilled in the art (e.g. adenosine diphosphate least one marker chosen from the group consisting of (a) (ADP), serotonin, collagen, protease, or ristocetine). Di- markers of inflammation, (b) markers of endothel func- agnosis can be performed by comparing the result of the tion, (c) markers of ischemia, (d) markers of thrombocyte 40 measurement to result of the same experiment in a con- activation, (e) markers of atherosclerosis activation, (f) trol sample or control group. Therefore, such test may markers of the transforming growth factor-β cytokine su- be considered a marker according to the present inven- perfamily, and (g) markers of intravascular activation of tion. coagulation. [0094] Markers of atherosclerosis activation according [0089] Measuring an additional marker may increase 45 to the present invention comprise any markers indicative the selectivity and specificity of diagnosis. It may also of progression of atherosclerosis. Examples for markers serve to confirm a diagnosis established by measuring of atherosclerosis activation include lipoprotein-associ- the level of VERY LOW CONCENTRATIONS OF TRO- ated phospholipase A2 (Lp-PLA2). PONIN I OR T. [0095] An example for markers of the transforming [0090] Markers of inflammation according to the 50 growth factor-β superfamily is the growth differentiation present invention include any markers indicative of an factor 15 (GDF-15), also known as MIC-1. inflammatory process, particularly a vascular or arterial [0096] Examples for markers of intravascular activa- inflammatory process. Particularly, markers of inflamma- tion of coagulation include fibrinogen degradation prod- tion according to the present invention comprise inflam- ucts, D-dimer, plasminogen activator inhibitor. matory active cytokines. Examples for markers of inflam- 55 [0097] Diagnosis according to the present invention is mation include interferons (e.g. interferon gamma), inter- preferably done by use of a diagnostic means. A diag- leukins (e.g. IL-1, IL-6, IL-8), Tumor necrosis factor (TNF) nostic means is any means that allows to measure the alpha), CRP (C-reactive protein), hsCRP (high-sensitiv- level amount, or concentration of a substance of interest,

13 25 EP 2 133 696 A1 26 particularly a peptide or polypeptide of interest, more par- [0105] Peptides and polypeptides (proteins) can be ticularly VERY LOW CONCENTRATIONS OF TRO- measured in tissue, cell, and body fluid samples, i.e. pref- PONIN I OR T. erably in vitro. Preferably, the peptide or polypeptide of [0098] Methods and diagnostic means which can be interest is measured in a body fluid sample. used to determine the levels of the respective peptides 5 [0106] A tissue sample according to the present inven- are known to the person skilled in the art. These methods tion refers to any kind of tissue obtained from the dead include microplate ELISA-based methods, fully-automat- or alive human or animal body. Tissue samples can be ed or robotic immunoassays (available for example on obtained by any method known to the person skilled in Elecsys™ analyzers), CBA (an enzymatic Cobalt Binding the art, for example by biopsy or curettage. Assay, available for example on Roche-Hitachi™ analyz- 10 [0107] Body fluids according to the present invention ers), and latex agglutination assays (available for exam- may include blood, blood serum, blood plasma, lymphe, ple on Roche-Hitachi™ analyzers). Furthermore, the per- cerebral liquor, saliva, and urine. Particularly, body fluids son skilled in the art is familiar with different methods of include blood, blood serum, blood plasma, and urine. measuring the level of a peptide or polypeptide. The term Samples of body fluids can be obtained by any method "level" relates to amount or concentration of a peptide or 15 known in the art. polypeptide in a patient (more specifically, in the blood [0108] Methods to obtain cell samples include directly or urine of the patient), or a sample taken from a patient preparing single cells or small cell groups, dissociating (e.g. a blood or urine sample). The term "measuring" ac- tissue (e.g. using trypsin), and separating cells from body cording to the present invention relates to determining fluids, e.g. by filtration or centrifugation. Cells according the amount or concentration, preferably semi-quantita- 20 to the present invention comprise also platelets and other tively or quantitatively, of the nucleic acid, peptide, non-nuclear cells, e.g. erythrocytes. polypeptide, or other substance of interest. Measuring [0109] If necessary, the samples may be further proc- can be done directly or indirectly. Indirect measuring in- essed. Particularly, nucleic acids, peptides or polypep- cludes measuring of cellular responses, bound ligands, tides may be purified from the sample according to meth- labels, or enzymatic reaction products. 25 ods known in the art, including filtration, centrifugation, [0099] In the context of the present invention, amount or extraction methods such as chloroform/phenol extrac- also relates to concentration. It is evident, that from the tion. total amount of a substance of interest in a sample of [0110] For measuring cellular responses, the sample known size, the concentration of the substance can be or processed sample is added to a cell culture and an calculated, and vice versa. 30 internal or external cellular response is measured. The [0100] Measuring can be done according to any meth- cellular response may include the expression of a report- od known in the art. Preferred methods are described in er gene or the secretion of a substance, e.g. a peptide, the following. polypeptide, or a small molecule. [0101] In one embodiment, the method for measuring [0111] Other preferred methods for measurement may the level of a peptide or polypeptide of interest, particu- 35 include measuring the amount of a ligand binding spe- larly VERY LOW CONCENTRATIONS OF TROPONIN cifically to the peptide or polypeptide of interest. Binding I OR T, comprises the steps of (a) contacting a cell ca- according to the present invention includes both covalent pable of a cellular response to the peptide or polypeptide and non-covalent binding. with the peptide or polypeptide for an adequate period [0112] A ligand according to the present invention can of time, (b) measuring the cellular response. 40 be any peptide, polypeptide, nucleic acid, or other sub- [0102] In another embodiment, the method for meas- stance binding to the peptide or polypeptide of interest. uring the level of a peptide or polypeptide of interest, It is well known that peptides or polypeptides, if obtained particularly VERY LOW CONCENTRATIONS OF TRO- or purified from the human or animal body, can be mod- PONIN I OR T, comprises the steps of (a) contacting a ified, e.g. by glycosylation. A suitable ligand according peptide or polypeptide with a suitable substrate for an 45 to the present invention may bind the peptide or polypep- adequate period of time, (b) measuring the amount of tide also via such sites. product [0113] Preferably, the ligand should bind specifically [0103] In another embodiment, the method for meas- to the peptide or polypeptide to be measured. "Specific uring the level of a peptide or polypeptide of interest, binding" according to the present invention means that particularly VERY LOW CONCENTRATIONS OF TRO- 50 the ligand should not bind substantially to ("cross-react" PONIN I OR T, comprises the steps of (a) contacting a with) another peptide, polypeptide or substance present peptide or polypeptide with a specifically binding ligand, in the sample investigated. Preferably, the specifically (b) (optionally) removing non-bound ligand, (c) measur- bound protein or isoform should be bound with at least ing the amount of bound ligand. 3 times higher, more preferably at least 10 times higher [0104] Preferably, the peptide or polypeptide is con- 55 and even more preferably at least 50 times higher affinity tained in a sample, particularly a body fluid or tissue sam- than any other relevant peptide or polypeptide. ple, and the amount of the peptide or polypeptide in the [0114] Non-specific binding may be tolerable, particu- sample is measured. larly if the investigated peptide or polypeptide can still be

14 27 EP 2 133 696 A1 28 distinguished and measured unequivocally, e.g. accord- strates for detection include di-amino-benzidine (DAB), ing to its size on a Western Blot, or by its relatively higher 3,3’-5,5’-tetramethylbenzidine, NBT-BCIP (4-nitro blue abundance in the sample. tetrazolium chloride and 5-bromo-4-chloro-3-indolyl- [0115] Binding of the ligand can be measured by any phosphate, available as ready-made stock solution from method known in the art. Preferably, the method is semi- 5 Roche Diagnostics), CDP-Star™ (Amersham Bioscienc- quantitative or quantitative. Suitable methods are de- es), ECF™ (Amersham Biosciences). A suitable en- scribed in the following. zyme-substrate combination may result in a colored re- [0116] First, binding of a ligand may be measured di- action product, fluorescence or chemoluminescence, rectly, e.g. by NMR or surface plasmon resonance. which can be measured according to methods known in [0117] Second, if the ligand also serves as a substrate 10 the art (e.g. using a light-sensitive film or a suitable cam- of an enzymatic activity of the peptide or polypeptide of era system). As for measuring the enyzmatic reaction, interest, an enzymatic reaction product may be meas- the criteria given above apply analogously. ured (e.g. the amount of a protease can be measured by [0124] Typical fluorescent labels include fluorescent measuring the amount of cleaved substrate, e.g. on a proteins (such as GFP and its derivatives), Cy3, Cy5, Western Blot). 15 Texas Red, Fluorescein, and the Alexa dyes (e.g. Alexa [0118] For measurement of enzymatic reaction prod- 568). Further fluorescent labels are available e.g. from ucts, preferably the amount of substrate is saturating. Molcular Probes (Oregon). Also the use of quantum dots The substrate may also be labeled with an detectable as fluorescent labels is contemplated. lable prior to the reaction. Preferably, the sample is con- [0125] Typical radioactive labels include 35S, 125I, tacted with the substrate for an adequate period of time. 20 32P, 33P and the like. A radioactive label can be detected An adequate period of time refers to the time necessary by any method known and appropriate, e.g. a light-sen- for a detectable, preferably measurable amount of prod- sitive film or a phosphor imager. uct to be produced. Instead of measuring the amount of [0126] Suitable measurement methods according the product, the time necessary for appearance of a given present invention also include precipitation (particularly (e.g. detectable) amount of product can be measured. 25 immunoprecipitation), electrochemiluminescence (elec- [0119] Third, the ligand may be coupled covalently or tro-generated chemiluminescence), RIA (radioimmu- non-covalently to a label allowing detection and meas- noassay), ELISA (enzyme-linked immunosorbent as- urement of the ligand. say), sandwich enzyme immune tests, electrochemilu- [0120] Labeling may be done by direct or indirect meth- minescence sandwich immunoassays (ECLIA), dissoci- ods. Direct labeling involves coupling of the label directly 30 ation-enhanced lanthanide fluoro immuno assay (DEL- (covalently or non-covalently) to the ligand. Indirect la- FIA), scintillation proximity assay (SPA), turbidimetry, beling involves binding (covalently or non-covalently) of nephelometry, latex-enhanced turbidimetry or neph- a secondary ligand to the first ligand. The secondary lig- elometry, or solid phase immune tests. Further methods and should specifically bind to the first ligand. Said sec- known in the art (such as gel electrophoresis, 2D gel ondary ligand may be coupled with a suitable label and/or 35 electrophoresis, SDS polyacrylamid gel electrophoresis be the target (receptor) of tertiary ligand binding to the (SDS-PAGE), Western Blotting, and mass spectrome- secondary ligand. The use of secondary, tertiary or even try), can be used alone or in combination with labeling or higher order ligands is often used to increase the signal. other dectection methods as described above. Suitable secondary and higher order ligands may include [0127] Preferred ligands include antibodies, nucleic antibodies, secondary antibodies, and the well-known 40 acids, peptides or polypeptides, and aptamers, e.g. nu- streptavidin-biotin system (Vector Laboratories, Inc.) cleic acid or peptide aptamers. Methods to such ligands [0121] The ligand or substrate may also be "tagged" are well-known in the art. For example, identification and with one or more tags as known in the art. Such tags may production of suitable antibodies or aptamers is also of- then be targets for higher order ligands. Suitable tags fered by commercial suppliers. The person skilled in the include biotin, digoxygenin, His-Tag, Glutathion-S- 45 art is familiar with methods to develop derivatives of such Transferase, FLAG, GFP, myc-tag, influenza A virus hae- ligands with higher affinity or specificity. For example, magglutinin (HA), maltose binding protein, and the like. random mutations can be introduced into the nucleic ac- In the case of a peptide or polypeptide, the tag is prefer- ids, peptides or polypeptides. These derivatives can then ably at the N-terminus and/or C-terminus. be tested for binding according to screening procedures [0122] Suitable labels are any labels detectable by an 50 known in the art, e.g. phage display. appropriate detection method. Typical labels include gold [0128] The term "antibody" as used herein includes particles, latex beads, acridan ester, luminol, ruthenium, both polyclonal and monoclonal antibodies, as well as enzymatically active labels, radioactive labels, magnetic fragments thereof, such as Fv, Fab and F(ab)2 fragments labels ("e.g. magnetic beads", including paramagnetic that are capable of binding antigen or hapten. The and superparamagnetic labels), and fluorescent labels. 55 present invention also includes "humanized" hybrid an- [0123] Enzymatically active labels include e.g. horse- tibodies wherein amino acid sequences of a non-human radish peroxidase, alkaline phosphatase, beta-Galactos- donor antibody exhibiting a desired antigen-specificity idase, Luciferase, and derivatives thereof. Suitable sub- are combined with sequences of a human acceptor an-

15 29 EP 2 133 696 A1 30 tibody. The donor sequences will usually include at least blood glucose measurement. Thus, a patient will be able the antigen-binding amino acid residues of the donor but to obtain the sample and measure the marker without may comprise other structurally and/or functionally rele- immediate assistance of a trained physician or nurse. vant amino acid residues of the donor antibody as well. [0137] Means suitable for measuring the expression Such hybrids can be prepared by several methods well 5 level of a marker according to the present invention, such known in the art. as antibodies, aptamers, antisense nucleic acids etc. [0129] In another preferred embodiment, the ligand, have been already been described in detail earlier in this preferably chosen from the group consisting of nucleic specification. In a preferred embodiment, the means is acids, peptides, polypeptides, more preferably from the packed as a kit comprising a container for the means or group consisting of nucleic acids, antibodies, or aptam- 10 agent for measurement as well as containers for any aux- ers, is present on an array. iliary agents for measurement, e.g. suitable buffers, fil- [0130] Said array contains at least one additional lig- ters, columns, enyzmes, etc.. and, which may be directed against a peptide, polypep- [0138] In another preferred embodiment, it is contem- tide or a nucleic acid of interest. Said additional ligand plated that the means is a lab-on-a-chip device or any may also be directed against a peptide, polypeptide or a 15 other device suitable for point-of-care diagnosis. Devices nucleic acid of no particular interest in the context of the for point-of-care diagnosis are known in the art. Prefer- present invention. Preferably, ligands for at least three, ably, such a device comprises a sampling unit (e.g. for preferably at least five, more preferably at least eight pep- taking a blood sample) and a measurement unit (e.g. for tides or polypeptides of interest in the context of the measuring the binding of the marker to a ligand). The present invention are contained on the array. 20 means may also be a test strip or other auxiliary tool for [0131] According to the present invention, the term "ar- measuring the expression level of the marker in such a ray" refers to a solid-phase or gel-like carrier upon which device. at least two compounds are attached or bound in one-, [0139] Therefore, in another embodiment, the present two- or three-dimensional arrangement. Such arrays (in- invention also relates to a kit, a lab-on-chip device, or a cluding "gene chips", "protein chips", antibody arrays and 25 point-of-care diagnostic device for measuring the expres- the like) are generally known to the person skilled in the sion level of the marker. art and typically generated on glass microscope slides, [0140] The method according to the present invention specially coated glass slides such as polycation-, nitro- comprises the step of diagnosing the risk of the patient cellulose- or biotin-coated slides, cover slips, and mem- by comparing the measured level to known levels asso- branes such as, for example, membranes based on ni- 30 ciated with different grades of risk in a patient. trocellulose or nylon. [0141] The person skilled in the art is able to determine [0132] The array may include a bound ligand or at least known levels of VERY LOW CONCENTRATIONS OF two cells expressing each at least one ligand. TROPONIN I OR T which are associated with different [0133] It is also contemplated to use "suspension ar- grades of cardiovascular risk with respect to administra- rays" as arrays according to the present invention (Nolan 35 tion of an anti-inflammatory drug, particularly an NSAID, JP, Sklar LA. (2002). Suspension array technology: ev- steroid, or selective Cox-2 inhibitor. In general, the higher olution of the flat-array paradigm. Trends Biotechnol. 20 the level of the VERY LOW CONCENTRATIONS OF (1):9-12). In such suspension arrays, the carrier, e.g. a TROPONIN I OR T, the higher is the risk for the patient. microbead or microsphere, is present in suspension. The [0142] According to the present invention, the term array consists of different microbeads or microspheres, 40 "risk" relates to the probability of a particular incident, possibly labeled, carrying different ligands. more particularly a cardiovascular complication, to take [0134] The invention further relates to a method of pro- place. The grade of risk can be increased or highly in- ducing arrays as defined above, wherein at least one creased. The grade of risk can also not be increased. ligand is bound to the carrier material in addition to other "No increased risk" means that there is apparently no ligands. 45 risk of a cardiovascular event with respect to administra- [0135] Methods of producing such arrays, for example tion of an anti-inflammatory drug, particularly an NSAID, based on solid-phase chemistry and photo-labile protec- steroid, or selective Cox-2 inhibitor. tive groups, are generally known (US 5,744,305). Such [0143] Guidance as to what levels are associated with arrays can also be brought into contact with substances which grade of risk can be drawn from levels of VERY or substance libraries and tested for interaction, for ex- 50 LOW CONCENTRATIONS OF TROPONIN I OR T ample for binding or change of confirmation. Therefore, known to be associated with the presence or severity of arrays comprising a peptide or polypeptide as defined a cardiovascular disease. For example, based on a 99. above may be used for identifying ligands binding spe- percentile obtained in individuals a plasma level of 12 cifically to said peptides or polypeptides. pg/ml of VERY LOW CONCENTRATIONS OF TRO- [0136] Furthermore, it is contemplated to use so called 55 PONIN T may be considered a normal level. Higher levels point-of-care or lab-on-a-chip devices for obtaining the of GDF-15 correlate for example with the level of symp- sample and measurement of the marker. Such devices toms according to the NYHA classification and with the may be designed analogously to the devices used in level of impairment of LVEF. The term "plasma level"

16 31 EP 2 133 696 A1 32 relates to levels of VERY LOW CONCENTRATIONS OF unnoticed cardiovascular risk in a patient. Thus, a patient TROPONIN I OR T measured in blood plasma. The levels having an increased or highly increased cardiovascular measured in plasma are generally comparable to the lev- risk is preferably subjected to further diagnosis to identify els measured in blood serum. an underlying cardiovascular disorder. This will allow to [0144] Below, plasma levels of VERY LOW CONCEN- 5 initiate treatment early, i.e. before the onset of obvious TRATIONS OF TROPONIN I OR T are given which are symptoms of the cardiovascular disorder. Thus, the gen- or can be typically considered to be associated with the eral health of the patient will profit from the present in- indicated grades of cardiovascular risk with respect to vention’s methods to diagnose the cardiovascular risk administration of an anti-inflammatory drug, a Cox-2 in- before or during administration of an anti-inflammatory hibiting compound, particularly an NSAID, steroid, or se- 10 drug. Furthermore, if treatment of the underlying cardio- lective Cox-2 inhibitor. vascular disorder is successful, the risk may be reduced [0145] Typically, a plasma level of less than 3,7 pg/ml (as diagnosed according to the means and methods pro- of TROPONIN T is associated with no increased cardi- vided by the present invention) and treatment with an ovascular risk with respect to administration of an anti- anti-inflammatory drug, particularly an NSAID, steroid, inflammatory drug, a Cox-2 inhibitor, particularly an 15 or selective Cox-2 inhibitor can be initiated or the dosage NSAID, steroid, or selective Cox-2 inhibitor. of such drug can be increased. [0146] Typically, a plasma level from 3,7 to 10 pg/ml [0151] Treatment of a patient having an increased risk of TROPONIN T is associated with an increased cardi- may also be accompanied by further measures such as ovascular risk with respect to administration of an anti- limiting or reducing the dosage of an anti-inflammatory inflammatory drug, a Cox-2 inhibitor, particularly an 20 drug administered or about to be administered, restriction NSAID, steroid, or selective Cox-2 inhibitor. However, of salt intake, regular moderate exercise, providing influ- also a level between 3,7 pg/ml and 12 pg/ml should war- enzal and pneumococcal immunization, surgical treat- rant further clarification of whether an increased risk is ment (e.g. revascularization, ballon dilatation, stenting, present. by-pass surgery), administering drugs such as diuretics [0147] Typically, a plasma level more than 10 pg/ml of 25 (including co-administration of more than one diuretic), TROPONIN T is associated with a highly increased car- ACE (angiotensin converting enzyme) inhibitors, β- diovascular risk with respect to administration of an anti- adrenergic blockers, aldosteron antagonists, calcium an- inflammatory drug, a Cox-2 inhibitor, particularly an tagonists (e.g. calcium channel blockers), angiotensin- NSAID, steroid, or selective Cox-2 inhibitor. However, receptor blockers, digitalis and any other measures also a level of more than 3,7 pg/ml should warrant further 30 known and deemed appropriate by the person skilled in clarification of whether a highly increased risk is present. the art. [0148] Once the risk in a patient has been diagnosed, [0152] The present invention also comprises a method it may have consequences for the subsequent treatment of monitoring the treatment with a Cox-2 inhibiting com- as described below. The grades of risk mentioned below pound, particularly an NSAID, steroid, or selective Cox- particularly refer to the grades of risk associated with the 35 2 inhibitor, wherein the level of TROPONIN I OR T is above described levels of TROPONIN I OR T. measured. [0149] If a method according to the present invention [0153] If, based upon consideration of risk and poten- indicates no increased risk, then the anti-inflammatory tial benefit, treatment with a Cox-2 inhibiting compound, drug may be administered, preferably taking into account in particular with an NSAID, steroid, or selective Cox-2 any other known risk factors for cardiovascular disease. 40 inhibitor is initiated, it may also be as an intermittent ther- Preferably, administration of the drug is accompanied by apy. Measuring TROPONIN I OR T may then be used to further monitoring of the level of the TROPONIN I OR T, monitor therapy, particularly intermittent therapy, and/or particularly in case of high dosage or long-term applica- to identify an elevation of the risk. The administration is tion of the drug. Thus, it will be possible to early detect stopped when the level of TROPONIN I OR T reaches a any unusual increase in the level of the TROPONIN I OR 45 certain value and is optionally re-initiated when the level T which would indicate an elevation of the cardiovascular falls below a certain value. The respective values of the risk. TROPONIN I OR T are those mentioned beforehand. For [0150] If a method according to the present invention example, a value indicating to stop administration may indicates an increased risk, then the patient is preferably be a value which normally would indicate a highly in- investigated intensively by further diagnosis according 50 creased risk (as described elsewhere in this specifica- to methods known to the skilled cardiologist, such as tion). Then, a value indicating to optionally re-initiate electrocardiography, echocardiography. Treatment with treatment may be a value indicating merely an increased a an anti-inflammatory drug, most particularly a selective risk (as described elsewhere in this specification) or a Cox-2 inhibitor, should only be initiated upon careful con- value indicating no increased risk (as described else- sideration of risk and potential benefit. Notably, the 55 where in this specification). present invention will not only help to make the adminis- [0154] From the above it is evident that the present tration of anti-inflammatory drugs safer by identifying risk invention also provides a method of monitoring of a pa- patients, but it may also help to uncover a previously tient who is being treated or who is about to be treated

17 33 EP 2 133 696 A1 34 with a Cox-2 inhibiting compound, in particular an NSAID, Example1 steroid, or a selective Cox-2 inhibitor. [0155] If a method according to the present invention [0160] A 70-year-old patient suffers from severe rheu- indicates a highly increased risk, then treatment may be matoid arthritis and has received long-term treatment adapted as described for increased risk. However, ad- 5 with ibuprofen. During the ibuprofen treatment he has ministration of a Cox-2 inhibiting compound, in particular suffered from gastrointestinal side-effects including gas- an NSAID, steroid, or a selective Cox-2 inhibitor, will a trointestinal bleeds. The physician considers to change priori not be a treatment option for a patient with a highly treatment to the use of selective Cox-2 inhibitors and increased risk. If, for whatever the reason may be, a Cox- performs a measurement of TROPONIN T. The Troponin 2 inhibiting compound, in particular an NSAID, or steroid, 10 T level is 15 pg/ml. The patient is referred to a cardiologist most particularly a selective Cox-2 inhibitor, is adminis- for further diagnosis. By means of echocardiogram, the tered, it should only be done under careful medical su- cardiologist diagnoses an ischemic function defect and pervision, in particular including measuring of TROPON- a risk for therapy with selective Cox-2 inhibitors. Since IN I OR T at short intervals to monitor the risk and/or to the cardiac function defect is not severe, an intermittent identify an elevation of the risk. 15 therapy with a selective Cox-2 inhibitor is initiated under close monitoring of TROPONIN T. Examples: Example 2 [0156] The following examples illustrate the invention and are not intended to limit its scope in any way. 20 [0161] A 55-year-old female patient has been treated for 6 years for diabetes type II and is suffering from a General chronic painful rheumatic disease. Therefore, her physi- cian considers treatment with Cox-2 inhibitors. The TRO- Measurement of TROPONIN T: PONIN T value is measured and determined to be 35 25 pg/ml. The patient is referred to a cardiologist for further [0157] TROPONIN T can be determined by an elec- diagnosis. The cardiologic examination shows that the trochemoluminescence immunoassay the Elecsys™ in- patient is suffering from congestive heart failure NYHA strument (Roche Diagnostics GmbH, Mannheim, Ger- class III with a left ventricular ejection fraction below 35%. many). The assay works according to the electrochem- Due to the high TROPONIN I OR T value and due to the oluminescence sandwich immunoassay principle. In a 30 co-morbidity of diabetes, treatment with Cox-2 inhibitors first step, the biotin-labelled IgG (1-21) capture antibody, is not initiated. the ruthenium-labelled F(ab’)2 (39-50) signal antibody and 20 microliters of sample are incubated at 37 °C for Example 3 9 minutes. Afterwards, streptavidin-coated magnetic mi- croparticles are added and the mixture is incubated for 35 [0162] A 72-year-old patient healthy and smoker, but additional 9 minutes. After the second incubation, the having no further traditional risk factors for heart disease, reaction mixture is transferred to the measuring cell of suffers from strong joint pain in the knees (probably due the system where the beats are magnetically captured to long-time sport activities). The patient knows that his onto the surface of an electrode. Unbound label is re- stomach is very sensitive. Therefore, treatment with se- moved by washing the measuring cell with buffer. 40 lective Cox-2 inhibitors is considered. The measured [0158] In the last step, voltage is applied to the elec- plasma TROPONIN I OR T value is determined to be 3,5 trode in the presence of a tri-propylamine containing buff- pg/ml. As the PROCAM score is below 40 and the only er and the resulting electrochemoluminescent signal is traditional risk factor is smoking, treatment with selective recorded by a photomultiplier. All reagents and samples Cox-2 inhibitors is initiated. are handled fully automatically by the Elecsys™ instru- 45 ment. Results are determined via a calibration curve which is instrument-specifically generated by 2-point cal- Claims ibration and a master curve provided via the reagent bar- code. The test is performed according to the instructions 1. A method for diagnosing the risk of a patient to suffer of the manufacturer. 50 from a cardiovascular complication as a conse- [0159] Blood for analysis may be sampled in EDTA- quence of administration of a Cox-2 inhibiting com- tubes containing 5000 U aprotinine (Trasylol, Bayer, Ger- pound, comprising the steps of many) and Lithium-Heparin-tubes (for clinical chemistry), as appropriate. Blood and urine samples are immediately a) measuring the level of TROPONIN I OR T, spun for 10 min. at 3400 rpm at 4 ° C. Supernatants are 55 b) diagnosing the risk of the patient by compar- stored at -80 °C until analysis. ing the measured level to known levels associ- ated with different grades of risk in a patient.

18 35 EP 2 133 696 A1 36

2. The method according to claim 1, wherein the patient binding ligand being an antibody or an aptamer is does not show obvious symptoms of a pre-existing used. cardiovascular disease, risk or complication. 11. A method of deciding whether to initiate treatment 3. The method according to claim 1 or 2, wherein a 5 of a patient with a compound having Cox-2 inhibiting plasma level of less than 3,7 pg/ml of TROPONIN I properties, which method comprises OR T is associated with no increased risk of suffering from a cardiovascular complication. a) measuring, in vitro, the level of TROPONIN I OR T in the patient, 4. The method according to claim 1 or 2, wherein a 10 b) diagnosing the risk of the patient by compar- plasma level of more than 3,7 pg/ml and less than ing the measured level with known level(s) as- 10 pg/ml of TROPONIN I OR T is associated with an sociated with different grades of risk in a patient increased risk of suffering from a cardiovascular c) wherein, complication. 15 ca) if the risk is diagnosed not to be in- 5. The method according to claim 1 or 2, wherein a creased, then initiation of treatment is rec- plasma level of more than 10 pg/ml of TROPONIN I ommended, and/or OR T is associated with a highly increased risk of cb) if the risk is diagnosed to be increased suffering from a cardiovascular complication. or highly increased, then refraining from the 20 treatment is recommended, 6. The method according to any of claims 1 to 5, where- in the method is carried out within the monitoring of optionally in consideration of the result of an exam- a therapy with a selective Cox-2 inhibitor, preferably ination of the patient by a cardiologist. wherein the method is for monitoring of an intermit- tent therapy with a selective Cox-2 inhibitor. 25 12. The method according to claim 11, wherein the com- pound is a selective Cox-2 inhibitor. 7. The method according to claim 6, wherein the ad- ministration with the selective Cox-2 inhibitor is 13. The method according to claim 11 or 12, wherein stopped when the level of TROPONIN I OR T reach- es a prescribed value and is optionally re-initiated 30 a) if the measured plasma or serum level of when the level falls below the prescribed value. TROPONIN I OR T is 3,7 pg/ml or higher, it is recommended not to treat the patient or to ap- 8. The method according to any of claims 1 to 7, where- prove treatment under cardiovascular monitor- in additionally the level(s) of at least one marker cho- ing and/or at low dosage of the selective Cox-2 sen from the group consisting of 35 inhibitor, and/or b) if the measured measured plasma or serum a) markers of inflammation level of TROPONIN I OR T is 10 pg/ml or higher, b) markers of endothel function then it is recommended not to treat the patient. c) markers of ischemia d) markers of thrombocyte activation 40 14. A method to determine whether treatment of a pa- e) markers of atherosclerosis activation tient with an anti-inflammatory drug may be initiated, f) markers of intravascular activation of coagu- said method comprising lation a) measuring the level of TROPONIN I OR T in is measured. 45 a plasma or serum sample from the patient, b) wherein a measured plasma or serum level 9. The method according to any of claims 1 to 8, where- of TROPONIN I OR T of 3,7 pg/L or higher, par- in the cardiovascular complication is coronary heart ticularly of 10 pg/L or higher, indicates that treat- disease, stable angina pectoris, acute coronary syn- ment should not be initiatied. drome, unstable angina pectoris, myocardial infarc- 50 tion, ST-elevated myocardial infarction, non ST-ele- 15. The method according to claim 14, wherein the anti- vated myocardial infarction, or stroke. inflammatory drug is a selective Cox-2 inhibitor.

10. The method according to any of claims 1 to 9, where- in the level of the TROPONIN I OR T is measured 55 using a specifically binding ligand, an array, a micro- fluidic device, a chemiluminescence analyzer, or a robotic device, preferably wherein a specifically

19 EP 2 133 696 A1

20 EP 2 133 696 A1

21 EP 2 133 696 A1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• WO 2006077265 A [0012] • WO 02083913 A [0045] • EP 0394819 A [0013] • EP 1577673 A1 [0045] • WO 02089657 A [0045] • US 5744305 A [0058] [0135]

Non-patent literature cited in the description

• Topol EJ. Failing the public health--rofecoxib, Merck, • Hamm et al. NEJM, vol. 327, 146-50 [0013] and the FDA. N Engl J Med, 2004, vol. 351, 1707-9 • Ohmann et al. NEJM, 1996, vol. 335, 1333-34 [0013] [0004] • Christenson et al. ClinChem, 1998, vol. 44, 494-501 • Bombardier C ; Laine L ; Reicin A ; Shapiro D ; [0013] Burgos-Vargas R ; Davis B ; Day R ; Ferraz MB ; • James et al. ClinChem, 2006, vol. 52, 6832-37 Hawkey CJ ; Hochberg MC. VIGOR Study Group. [0013] Comparison of upper gastrointestinal toxicity of ro- • Latini et al. Circulation, 2007, vol. 116, 1242-49 fecoxib and naproxen in patients with rheumatoid ar- [0014] thritis. VIGOR Study Group. N Engl J Med, 2000, vol. • Nolan. Trends Biotechnol., 2002, vol. 20 (1), 9-12 343, 1520-8 [0004] [0058] • Jüni P ; Nartey L ; Reichenbach S ; Sterchi R ; •Warner, T.D.; Giuliano, F. ; Vojnovic, I. et al. Non- Dieppe PA ; Egger M. Risk of cardiovascular events steroid drug selectivities for cyclo-oxygenase-1 rath- and rofecoxib: cumulative meta-analysis. Lancet, er than cyclo-oxygenase-2 are associated with hu- 2004 [0004] man gastrointestinal toxicity: A full in vitro analysis. • Fitzgerald GA. Coxibs and cardiovascular disease. Proceedings of the National Academy of Sciences N Engl J Med, 2004, vol. 351, 1709-1711 [0005] USA, 1999, vol. 96, 7563-7568 [0079] • Mukherjee D ; Nissen SE ; Topol EJ. Risk of car- • Nolan JP ; Sklar LA. Suspension array technology: diovascular events associated with selective COX-2 evolution of the flat-array paradigm. Trends Biotech- Inhibitors. JAMA, 2001, vol. 286, 954-959 [0005] nol., 2002, vol. 20 (1), 9-12 [0133] • Katus et al. Mol Cell Cardiol, 1989, vol. 21, 1349-53 [0013]

22