U.S. patent application number 14/664243 was filed with the patent office on 2015-07-09 for methods of diagnosis and risk stratification of adverse events in post myocardial infarction patients using pro-adrenomedullin.
This patent application is currently assigned to BRAHMS GMBH. The applicant listed for this patent is BRAHMS GMBH. Invention is credited to ANDREAS BERGMANN, NILS MORGENTHALER, LEONG L. NG, JANA PAPASSOTIRIOU, JOACHIM STRUCK.
Application Number | 20150192595 14/664243 |
Document ID | / |
Family ID | 39733336 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150192595 |
Kind Code |
A1 |
NG; LEONG L. ; et
al. |
July 9, 2015 |
METHODS OF DIAGNOSIS AND RISK STRATIFICATION OF ADVERSE EVENTS IN
POST MYOCARDIAL INFARCTION PATIENTS USING PRO-ADRENOMEDULLIN
Abstract
The invention provides methods for the diagnosis and risk
stratification of adverse events in post-myocardial infarction
patients by means of proADM, whereby a determination of the marker
pro-adrenomedullin or partial sequence or a fragment thereof or
contained in a marker combination (panel, cluster) is carried out
on a post-myocardial infarction patient. The invention also
provides a diagnostic device and a kit for the performance of the
method of the method of the invention.
Inventors: |
NG; LEONG L.; (LEICESTER,
GB) ; BERGMANN; ANDREAS; (BERLIN, DE) ;
STRUCK; JOACHIM; (BERLIN, DE) ; MORGENTHALER;
NILS; (BERLIN, DE) ; PAPASSOTIRIOU; JANA;
(BERLIN, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRAHMS GMBH |
HENNIGSDORF |
|
DE |
|
|
Assignee: |
BRAHMS GMBH
HENNIGSDORF
DE
|
Family ID: |
39733336 |
Appl. No.: |
14/664243 |
Filed: |
March 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11937061 |
Nov 8, 2007 |
9012151 |
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14664243 |
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60858083 |
Nov 9, 2006 |
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Current U.S.
Class: |
436/501 |
Current CPC
Class: |
G01N 2800/50 20130101;
G01N 2800/324 20130101; G01N 33/74 20130101; G01N 33/6893
20130101 |
International
Class: |
G01N 33/74 20060101
G01N033/74 |
Claims
1. A method for the in vitro diagnosis and/or risk stratification
of an adverse event in post-myocardial infarction patients,
comprising the step of determining the level of pro-adrenomedullin
or partial peptides or fragments thereof from a post-myocardial
infarction patient, wherein a significantly elevated level of
pro-adrenomedullin correlates with a greater risk of an adverse
event in said post-myocardial infarction patient.
2. The method according to claim 1, wherein the fragment of
pro-adrenomedullin is MR-proADM (SEQ ID No. 2).
3. The method according to claim 1, wherein said adverse event is
at least one of myocardial infarction, heart failure and death.
4. The method of claim 1 further comprising the step of determining
the level of at least one marker selected from the group consisting
of BNP, proBNP, NT-proBNP or a partial sequence thereof in each
case, from said post-myocardial infarction patient.
5. The method according to claim 1 further comprising the step of
determining at least one marker selected from the group consisting
of inflammatory markers, cardiovascular markers, neurohormonal
markers or ischemic markers from said post-myocardial infarction
patient.
6. The method according to claim 5, wherein said inflammatory
marker is at least one marker selected form the group consisting of
C-reactive protein (CRP), procalcitonin (1-116, 3-116), cytokines,
interleukins, and adhesion molecules.
7. The method according to claim 6 wherein said cytokine is TNF
alpha (tumor necrosis factor-alpha); said interleukin is IL-6, or
said adhesion molecule is VCAM or ICAM.
8. The method according to claim 5, wherein said cardiovascular
marker is at least one marker selected from the group consisting
creatine kinase, myeloperoxidase, myoglobin, natriuretic proteins,
cardial troponin, CRP, and circulation-regulating
(pro)hormones.
9. The method according to claim 8 wherein said natriuretic protein
is selected from the group consisting of ANP, ANF, proANP,
NT-proANP, BNP, proBNP, NT-proBNP and partial sequences thereof in
each case.
10. The method according to claim 8 wherein said
circulation-regulating (pro)hormone is selected from the group
consisting of pro-gastrin-releasing peptide (proGRP),
pro-endothelin-1, pro-leptin, pro-neuropeptide-Y, pro-somatostatin,
pro-neuropeptide-YY, pro-opiomelanocortin and partial sequence
thereof in each case.
11. The method according to claim 5, wherein said ischemic marker
is at least one marker selected from the group consisting of
troponin I and T, and CK-MB.
12. The method according to claim 5, wherein said neurohormonal
marker is at least one natriuretic protein.
13. The method according to claim 12, wherein said at least one
natrriuretic protein is selected from the group consisting of ANP,
ANF, proANP, NT-proANP, BNP, proBNP (NT-proBNP) and partial
sequences thereof in each case.
14. The method according to claim 4 wherein parallel or
simultaneous determinations of the markers are carried out.
15. The method according to claim 5 wherein parallel or
simultaneous determinations of the markers are carried out.
16. The method according to claim 1, wherein the determination is
made in at least one specimen from said post-myocardial infarction
patient.
17. The method of claim 16 wherein said specimen is a blood
specimen.
18. A kit for the in vitro diagnosis and/or risk stratification of
an adverse event in post-myocardial infarction patients, containing
detection reagents for the determination of the marker
pro-adrenomedullin or partial peptides or fragments thereof or
contained in a marker combination, whereby the marker combination
can contain additional markers as well as an auxiliary agent.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/937,061 filed Nov. 8, 2007, which claims the benefit of U.S.
Provisional Application No. 60/858,083 filed Nov. 9, 2006. The
entire contents of each of these applications are hereby
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to a method for the diagnosis and/or
risk stratification of adverse events in post-myocardial infarction
patients, whereby a determination of the marker pro-adrenomedullin
(proADM) or a partial sequence or fragment thereof or contained in
a marker combination (panel, cluster) is carried out on a patient
who is to be examined.
BACKGROUND OF THE INVENTION
[0003] The diagnosis and risk stratification of adverse events
(death, heart attack, heart failure) in post-myocardial infarction
patients have been described in conjunction with natriuretic
proteins, namely, BNP and NTproBNP (Richards A. M., Nicholls M. G.,
Yandle T. G., Frampton C., Espiner E. A., Turner J. G., Buttimore
R. C., Lainchbury J. G., Elliott J. M., Ikram H., Crozier I. G.,
Smyth D. W. Plasma N-terminal pro-brain natriuretic peptide and
adrenomedullin: new neurohormonal predictors of left ventricular
function and prognosis after myocardial infarction. Circulation
1998; 97:1921-1929; Squire I. B., O'Brien R. J., Demme B., Davies
J. E., Ng L. L. N-terminal pro-atrial natriuretic peptide (N-ANP)
and N-terminal pro-B-type natriuretic peptide (N-BNP) in the
prediction of death and heart failure in unselected patients
following acute myocardial infarction. Clin. Sci. (London) 2004;
107:309-316). However, it is particularly difficult to make a
reliable diagnosis or to undertake a stratification in such
post-myocardial infarction patients, especially as far as further
clinical decisions are concerned.
[0004] Moreover, the state of the art describes pro-adrenomedullin
(proADM) determination within the scope of the diagnosis (EP
0622458 B1), particularly for purposes of examining sepsis (EP
1121600 B1).
[0005] Circulating natriuretic peptide levels such as N-terminal
pro B type natriuretic peptide (NTproBNP) provide prognostic
information in patients at predicting death and heart failure in
the post acute myocardial infarction (AMI) phase..sup.1 The
challenge remains to try and identify those patients who are deemed
to be at high risk of adverse events. The addition of biomarkers in
risk stratification has been shown to be superior to clinical
features at predicting adverse outcomes and this has also been
borne out in a range of acute coronary syndromes..sup.2 Newer
peptides are emerging which may give complementary and additional
information, particularly in a multi-marker strategy with NTproBNP.
Adrenomedullin (ADM) is a 52 amino acid peptide which has homology
with calcitonin gene related peptide..sup.3 It was originally
isolated from human pheochromocytoma cells, however it has been
detected in other tissues including adrenal medulla, heart, brain,
lung, kidney, and gastrointestinal organs.sup.3,4 and its mRNA is
highly expressed in endothelial cells..sup.5 The downstream actions
of ADM are mediated by an increase in cAMP levels..sup.6
[0006] ADM is synthesized as part of a larger precursor molecule,
termed preproadrenomedullin. In humans this precursor consists of
185 amino acids..sup.7 The gene encoding preproadrenomedullin is
termed the ADM gene and has been mapped and localized to chromosome
11..sup.8 ADM is difficult to measure in plasma as it can act in an
autocrine or paracrine way, is partially complexed with complement
factor H, and is rapidly cleared from the circulation..sup.9
Recently, the more stable midregional fragment of
pro-adrenomedullin (MR-proADM), comprising amino acids 45-92 of
preproADM, has been identified which is more stable than the active
molecule being secreted in equimolar amounts to
adrenomedullin..sup.10
[0007] The biological activity of ADM in the cardiovascular system
is similar to that of B-type natriuretic peptide (BNP) causing
vasodilation.sup.11 via production of NO.sup.12 increasing cardiac
output.sup.13 and inducing diuresis and natriuresis..sup.14 Plasma
ADM is increased in heart failure, in proportion to the severity of
disease.sup.15, 16 and is inversely related to LVEF.
[0008] Plasma ADM has been investigated previously in two small
studies as a prognostic marker comparing it to NTproBNP and
BNP..sup.1, 17 One study identified plasma ADM as an independent
predictor of cardiogenic shock and short term mortality.sup.17,
whereas ADM had no independent additional prognostic value to
NTproBNP in another.sup.1.
[0009] It is an object of the present invention to provide an
improved method for the diagnosis and risk stratification of
adverse events in post-myocardial infarction patients.
SUMMARY OF THE INVENTION
[0010] This objective is achieved by a method for in-vitro
diagnosis and/or risk stratification of adverse events in
post-myocardial infarction patients, whereby a determination of the
marker pro-adrenomedullin or a partial sequence or fragment thereof
or contained in a marker combination (panel, cluster) is carried
out on a patient who is to be examined.
[0011] Thus, one embodiment of the invention provides a method for
the in vitro diagnosis and/or risk stratification of an adverse
event in post-myocardial infarction patients, comprising the step
of determining the level of pro-adrenomedullin or partial peptides
or fragments thereof from a post-myocardial infarction patient,
wherein a significantly elevated level of pro-adrenomedullin
correlates with a greater risk of an adverse event in said
post-myocardial infarction patient. Preferably, the method
determines the level of a fragment of pro-adrenomedullin, MR-proADM
(SEQ ID No. 2). In preferred embodiments of the invention, adverse
event is at least one of myocardial infarction, heart failure and
death. The method of the invention can further comprise the step of
determining the level of at least one marker selected from the
group consisting of BNP, proBNP, NT-proBNP or a partial sequence
thereof in each case, from the post-myocardial infarction patient.
The method of the invention can also further comprise the step of
determining the level of at least one marker selected from the
group consisting of inflammatory markers, cardiovascular markers,
neurohormonal markers or ischemic markers from said post-myocardial
infarction patient.
[0012] Preferably, the determination is made in at least one
specimen from the post-myocardial infarction patient.
[0013] Another embodiment of the invention provides a kit for the
in vitro diagnosis and/or risk stratification of an adverse event
in post-myocardial infarction patients, containing detection
reagents for the determination of the marker pro-adrenomedullin or
partial peptides or fragments thereof or contained in a marker
combination, whereby the marker combination can contain additional
markers as well as an auxiliary agent.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows the amino acid sequence of preproADM (amino
acids 1-185); proADM (amino acids 22-185; 164 amino acids) and ADM
(amino acids 95-146; 52 amino acids).
[0015] FIG. 2 shows the amino acid sequence of mid-regional
pro-adrenomedullin (MR-proADM), which corresponds to amino acids
45-92 of preproADM shown in SEQ ID NO: 1.
[0016] FIG. 3 shows a combined Receiver Operating Characteristic
Curve comparing NTproBNP, MR-proADM and the combined predicted
probabilities from a binary logistic model for prediction of death
or heart failure.
[0017] FIG. 4 shows a Kaplan-Meier Curve: Time to death or heart
failure related to plasma MR-proADM.
[0018] FIG. 5 shows a Kaplan-Meier Curve: Time to death or heart
failure related to plasma NTproBNP.
[0019] FIG. 6 shows a Kaplan-Meier analysis for quartiles of
MR-proADM predicting the primary endpoint of death or heart
failure, in patients stratified by NTproBNP (<or>
median).
[0020] FIG. 7 shows annual event rates for death and for death or
heart failure, in patients stratified by NTproBNP (<or>
median) and MRproADM quartiles.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The role of the more stable prohormone MRproADM in the
prognostication of AMI was unknown prior to the studies of the
present invention, in which the inventors investigated whether
MR-proADM would be of benefit in determining the prognosis
following AMI, particularly for predicting death and heart failure.
The results were compared with NTproBNP, a peptide of established
prognostic value in this group of patients..sup.1,18,19
[0022] According to the invention, the term "risk stratification"
comprises the identification of post-myocardial infarction
patients, especially emergency patients and risk patients, who
display a worse prognosis, for purposes of obtaining a more
thorough diagnosis and (follow-up) therapy or treatment with the
objective of attaining the most favorable possible outcome for the
heart disease.
[0023] Consequently, especially in cases of emergency and/or
intensive medical care, the method according to the invention is
particularly advantageous for obtaining a reliable diagnosis and/or
risk stratification. The method according to the invention allows
clinical decisions that lead to a fast therapy success. Such
clinical decisions likewise comprise further treatment by means of
drugs for the treatment or therapy of myocardial infarction or
heart failure.
[0024] In another preferred embodiment of the method according to
the invention, the diagnosis and/or risk stratification is carried
out to obtain a prognosis, a prophylaxis, a differential-diagnostic
early detection and recognition, an evaluation of the severity and
an outcome assessment of an adverse event in post-myocardial
infarction patients during their therapy.
[0025] In another embodiment of the method according to the
invention, blood--optionally whole blood, serum or available
plasma--is drawn from the patient who is to be examined and the
diagnosis is made in vitro/ex vivo, that is to say, outside of the
human or animal body. The diagnosis and/or risk stratification can
be made on the basis of the determination of the marker
pro-adrenomedullin or partial peptides or fragments thereof and the
amount present in at least one specimen from the patient.
[0026] Within the scope of this invention, the term "adverse
(negative) event in post-myocardial infarction patients" refers
especially to another (subsequent) myocardial infarction, heart
failure or the occurrence of death or other deterioration of the
prognosis of the patient.
[0027] Within the scope of this invention, the term "myocardial
infarction" (heart attack, acute myocardial infarction--AMI) refers
to acute and life-threatening heart disease, whereby necrosis or
tissue death (infarction) of parts of the heart muscle (myocardium)
occur due to impaired perfusion (ischemia) lasting, as a rule, more
than 20 minutes. The cardinal symptom of heart attack is the sudden
occurrence of usually strong pain in the chest area (chest pain)
that lasts more than 20 minutes and that can radiate into the
shoulders, arms, lower jaw and epigastric region and that can be
accompanied by sweating, nausea and possibly vomiting. Heart
failure is possible as a result of a myocardial infarction.
[0028] The term "post-myocardial infarction patient" means that
this patient has already suffered a myocardial infarction in the
past, that is to say, for instance, more than 1 hour, especially
more than 20 hours, especially 1 to 5 days or 3 to 5 days ago, and
is now in the post-phase and who did not die immediately, but for
whom another adverse event can be expected either directly or
indirectly.
[0029] All of the above-mentioned indications are also described,
for example, in Pschyrembel, published by de Gruyter, Berlin,
Germany 2004.
[0030] Within the scope of this invention, the term
"pro-adrenomedullin (proADM)" refers to a human protein or
polypeptide with an amino acid sequence of 22-185 (position 22 is
Ala, position 185 is Leu) of SEQ ID No. 1 (FIG. 1) of the
pre-pro-adrenomedullin (Kitamura K., Sakata J., Kangawa K., Kojima
M., Matsuo H., Eto T. Cloning and characterization of cDNA encoding
a precursor for human adrenomedullin, Biochem. Biophys. Res.
Commun. 1993; 194:720-725) as well as its fragments and partial
peptides, especially an amino acid sequence of 95-146 (position 95
is Tyr and position 146 is Tyr) of SEQ ID No. 1 (FIG. 1) of the
pre-pro-adrenomedullin, namely, adrenomedullin or an amino acid
sequence of 45-92 (position 45 is Glu and position 92 is Val) of
SEQ ID No. 1 (FIG. 1) of pre-pro-adrenomedullin or amino acid
sequence 1-48 of SEQ ID No. 2 (FIG. 2). This fragment is also
referred to as mid-regional pro-adrenomedullin (MR-proADM) (EP
1488209 B1) and, according to the invention, it constitutes an
especially preferred fragment because of its high plasma stability.
Likewise preferred are those fragments of pro-adrenomedullin that
have an N-terminal cleavage of amino acids (so-called NT-proADM),
whereby the first 5, 10, 15 or 20 amino acids of the proADM of the
N-terminus (starting at position 22 of SEQ ID No. 1) have been
deleted (also see EP 0622458 B1). Therefore, partial peptides
according to the invention can be the pro-adrenomedullin N-terminal
20-peptide (PAMP), which exhibits hypotensive properties, that is
to say, it lowers the blood pressure. A less thoroughly studied
C-terminal partial peptide--referred to as adrenotensin--consisting
of 33 amino acids (amino acids 153-185 of the preproADM (SEQ ID No.
1)) is encompassed according to the invention. Moreover, the
pro-adrenomedullin according to the invention can exhibit
posttranslational modifications such as glycolization,
lip(o)idization or derivatizations.
[0031] In another embodiment, the determination of
pro-adrenomedullin can be additionally carried out with other
markers, whereby pro-adrenomedullin is contained in a marker
combination (panel, cluster), namely, preferably those that already
indicate heart disease, especially myocardial infarction.
[0032] For this reason, the invention relates to an embodiment of
the method according to the invention in which the determination is
additionally carried out on a patient who is to be examined with at
least one additional marker selected from the group of inflammatory
markers, cardiovascular markers, neurohormonal markers or ischemic
markers.
[0033] According to the invention, the inflammatory marker can be
selected from at least one marker from the group of C-reactive
protein (CRP), cytokines such as, for instance, TNF alpha (tumor
necrosis factor-alpha), interleukins such as IL-6, procalcitonin
(1-116, 3-116) and adhesion molecules such as VCAM or ICAM, as well
as the cardiovascular markers from at least one marker selected
from the group consisting of creatine kinase, myeloperoxidase,
myoglobin, natriuretic protein, especially ANP (or ANF), proANP,
NT-proANP, BNP, proBNP, NT-proBNP or a partial sequence thereof in
each case, cardial troponin, CRP. Moreover, this also refers to
circulation-regulating (pro)hormones, particularly such as
pro-gastrin-releasing peptide (proGRP), pro-endothelin-1,
pro-leptin, pro-neuropeptide-Y, pro-somatostatin,
pro-neuropeptide-YY, pro-opiomelanocortin or a partial sequence
thereof in each case.
[0034] The ischemic marker can be selected from at least one marker
from the group consisting of troponin I and T, CK-MB. Moreover, the
neurohormonal marker can be at least one natriuretic protein,
especially ANP (or ANF), proANP, NT-proANP, BNP, proBNP, NT-proBNP
or a partial sequence thereof in each case.
[0035] In a very especially preferred embodiment, the additional
marker is BNP, proBNP, NT-proBNP (for markers associated with BNP,
see: Tateyama et al., Biochem. Biophys. Res. Commun. 185: 760-7
(1992); Hunt et al., Biochem. Biophys. Res. Commun. 214: 1175-83
(1995)) or a partial sequence thereof in each case.
[0036] Particularly in the examples, it is demonstrated that a
marker combination of the marker according to the invention,
namely, pro-adrenomedullin, and NT-proBNP, has greater significance
for the diagnosis and/or risk stratification of post-myocardial
infarction patients in terms of an adverse event (outcome) at the
given endpoints.
[0037] In another embodiment of the invention, this preferred
combination, in turn, can be augmented by additional markers
mentioned here to form a marker combination (panel, cluster).
[0038] In another embodiment of the invention, the method according
to the invention can be carried out by means of parallel or
simultaneous determinations of the markers (e.g. multititer plates
with 96 or more cavities), whereby the determinations are made in
at least one specimen from a patient.
[0039] Moreover, the method according to the invention and its
determinations can be carried out in an automatic analysis device,
especially by means of a Kryptor (http://www.kryptor.net/).
[0040] In another embodiment, the method according to the invention
and its determinations can be carried out by means of a quick test
(for example, lateral-flow test), whether it is in a
single-parameter or multi-parameter determination.
[0041] Moreover, the invention relates to the use of
pro-adrenomedullin or partial peptides or fragments thereof or
contained in a marker combination (panel, cluster) for the in vitro
diagnosis and/or risk stratification of an adverse event in
post-myocardial infarction patients, especially taking into
consideration the above-mentioned embodiments.
[0042] Another objective is to provide a corresponding diagnostic
device or the use of such a device for carrying out the method
according to the invention. Within the scope of this invention,
such a diagnostic device refers to an array or assay (for instance,
immunoassay, ELISA, etc.), in the broadest sense, to a device for
carrying out the method according to the invention.
[0043] The invention also relates to a kit or to the use of such a
kit for in vitro diagnosis and risk stratification of an adverse
event in post-myocardial infarction patients, whereby a
determination of pro-adrenomedullin or partial peptides or
fragments thereof or contained in a marker combination (panel,
cluster) is carried out on a patient who is to be examined,
especially taking into consideration the above-mentioned
embodiments. Such detection reagents comprise, for instance,
antibodies, etc.
[0044] The examples and figures below serve to elucidate the
invention in greater detail without, however, limiting the
invention to these examples and figures.
EXAMPLE
Study Population
[0045] The study population was 983 consecutive acute myocardial
infarction patients admitted to the Coronary Care Unit of Leicester
Royal Infirmary. The study complied with the Declaration of
Helsinki and was approved by the local ethics committee; written
informed consent was obtained from patients. AMI was defined at
presentation with at least two of three standard criteria, i.e.
appropriate symptoms, acute ECG changes of infarction (ST elevation
or depression, new left bundle branch block) and a rise in troponin
T above the 99.sup.th percentile for our population. AMI was
sub-categorised into ST segment elevation myocardial infarction
(STEMI) or non-ST segment myocardial infarction (NSTEMI). Exclusion
criteria were known malignancy, or surgery in the previous
month.
Plasma Samples
[0046] Blood samples were drawn at 3 to 5 days after the onset of
chest pain for determination of plasma MR-proADM and NTproBNP.
After 15 minutes bed rest, 20 mL blood was collected into tubes
containing EDTA and aprotinin. All plasma was stored at -70.degree.
C. until assayed in a blinded fashion in a single batch.
Echocardiography
[0047] Transthoracic echocardiography was performed in patients
using a Sonos 5500 instrument (Philips Medical Systems, Reigate,
UK). A 16-segment left ventricular wall motion index (LVWMI) based
on the American Society of Echocardiography mode was derived by
scoring each LV segment (1=normal, 2=hypokinesis, 3=akinesis and
4=dyskinesis (Paradoxical Motion), and dividing the total by the
number of segments scored. Left ventricular ejection fraction
(LVEF) was calculated using the biplane method of discs
formula..sup.20 Impaired LV systolic function was defined as an
EF<40% or a LVWMI>1.8.
NTproBNP Assay
[0048] The NTproBNP assay was based on a non-competitive assay as
previously published..sup.2 Sheep antibodies were raised to the
N-terminal of human NTproBNP and monoclonal mouse antibodies were
raised to the C-terminal. Antibodies raised against the N-terminal
were affinity-purified and biotinylated. Samples or NTproBNP
standards were incubated in C-terminal IgG-coated wells with the
biotinylated N-terminal antibody for 24 hours at 4.degree. C.
Detection was with methyl-acridinium ester (MAE)-labelled
streptavidin on a MLX plate luminometer (Dynex Technologies Ltd.,
Worthing, UK). The lower limit of detection was 0.3 pmol/L. There
was no cross reactivity with atrial natriuretic peptide, BNP, or
C-type natriuretic peptide.
MR-proADM Assay
[0049] MR-proADM was detected using a novel commercial assay in the
chemiluminescence/coated tube-format (BRAHMS A G) as described in
Morgenthaler N G, Struck J, Alonso C, Bergmann A. Measurement of
midregional proadrenomedullin in plasma with an immunoluminometric
assay. Clin Chem. 51(10):1823-9, Oct. 2005). Briefly, tubes were
coated with a purified sheep polyclonal antibody raised against a
peptide representing amino acids 83-94 of preproADM. A purified
sheep polyclonal antibody raised against a peptide representing
amino acids 68-86 of preproADM was labelled with
MACN-Acridinium-NHS-Ester (InVent GmbH, Germany) and used as
tracer. Dilutions of a peptide representing amino acids 45-92 of
preproADM in normal horse serum served as standards. The
immunoassay was performed by incubating 10 .mu.l of
samples/standards and 200 .mu.l tracer in coated tubes for 2 h at
room temperature. Tubes were washed 4 times with 1 ml of LIA wash
solution (BRAHMS A G), and bound chemiluminescence was measured
using a LB952T luminometer (Berthold, Germany).
End Points
[0050] The value of both MR-proADM and NTproBNP were assessed for
the prediction of death and heart failure. The combined primary
endpoint consisted of death and heart failure. Death, heart failure
and recurrent AMI were also investigated as individual secondary
endpoints. Myocardial infarction (MI) was diagnosed if a patient
had chest pain lasting >20 minutes, diagnostic serial
electrocardiographic (ECG) changes consisting of new pathological Q
waves or ST-segment and T-wave changes, and a plasma creatine
kinase-MB elevation greater than twice normal or cardiac troponin I
level>0.1 ng/mL..sup.21 Hospitalization for heart failure was
defined as a hospital admission for which heart failure was the
primary reason. Endpoints were obtained by reviewing the Office of
National Statistics Registry and by contacting each patient. There
was a minimum 30-day follow-up of all surviving patients.
Statistical Analysis
[0051] Statistical analyses were performed on SPSS Version 12 (SPSS
Inc, Chicago, Ill.). The continuous variables in the two
independent groups were compared using the Mann Whitney U test. To
test the independent predictive power for death or heart failure of
peptides levels above and below the median, Spearman's correlations
were performed and binary logistic regression analyses were
conducted. Baseline patient characteristics (age, sex, serum
creatinine, Killip class, and territory of AMI) and peptide markers
(including troponin I) were included as variables. Levels of
NTproBNP and MR-proADM were normalised by log transformation. Thus,
odds ratios and hazard ratios refer to a tenfold rise in the levels
of these markers.
[0052] To compare the predictive value of NTproBNP, MR-proADM or
the predicted probability derived from logistic regression
analyses, receiver-operating characteristic (ROC) curves were
generated and the area under the curves (AUC) was calculated. To
identify the independent predictors of death or heart failure, Cox
proportional hazard analyses was used. Kaplan Meier survival curves
were generated to visualise the relationship between the peptides
NTproBNP and MR-proADM and the primary and secondary endpoints. A p
value of less than 0.05 was deemed to be statistically
significant.
RESULTS
Patient Characteristics
[0053] The demographic features of the patient population are shown
in Table 1. Median length of follow-up was 342 days with a range of
0-764 days. No patient was lost to follow-up. During follow-up, 101
(10.3%) patients died and 49 (5.0%) were readmitted with heart
failure. There were 784 STEMI patients, 67.8% of whom were
thrombolysed. Echocardiographic data was available for 645 (65.6%)
of the 983 patients and done during index admission.
TABLE-US-00001 TABLE 1 Characteristics of the 983 patients in the
study separated by MR-proADM quartiles. Values are means (SD) or
numbers (%) 1st quartile 2nd quartile 3rd quartile 4th quartile p
value Age (in years) 55.5 .+-. 10.7 63.5 .+-. 10.1 67.4 .+-. 10.3
73.6 .+-. 10.1 <0.001 Previous Medical History AMI 25 (10.2) 37
(15.0) 43 (17.5) 59 (24.1) <0.0001 Angina Pectoris 52 (21.1) 57
(23.2) 68 (27.6) 72 (29.4) 0.150 Hypertension 80 (32.5) 105 (42.7)
108 (43.9) 126 (51.4) <0.0001 Diabetes mellitus 31 (12.6) 53
(21.5) 43 (17.5) 88 (35.9) <0.001 High cholesterol 51 (20.7) 56
(22.8) 59 (24.0) 58 (23.7) 0.843 Current/Ex-Smokers 166 (67.5) 153
(62.2) 146 (59.3) 140 (57.1) 0.06 ST-elevation AMI 187 (76.0) 200
(81.3) 205 (83.3) 201 (82.0) 0.244 Thrombolytic 136 (55.3) 131
(53.3) 146 (59.3) 111 (45.3) 0.043 Territory of Infarct 0.320
Anterior 108 (43.9) 106 (43.1) 95 (38.6) 101 (41.2) Inferior 100
(40.7) 86 (35.0) 103 (41.9) 85 (34.7) Other 37 (15.1) 54 (22.0) 48
(19.5) 60 (24.5) Killip Class on <0.001 Admission I 165 (67.0)
134 (54.5) 114 (46.3) 74 (30.2) II 63 (25.6) 94 (38.2) 104 (42.3)
114 (46.5) III 10 (4.1) 9 (3.7) 22 (8.9) 47 (19.2) IV 1 (0.4) 0 (0)
0 (0) 9 (3.7) Peak CK (IU/L) 955.1 .+-. 1054.6 1041.9 .+-. 1152.9
1063.8 .+-. 1124.3 1210.7 .+-. 1427.9 0.142 Creatinine (.mu.mol/L)
91.0 .+-. 17.6 91.3 .+-. 18.2 101.9 .+-. 26.0 125.6 .+-. 48.9
<0.001 NTproBNP (pmol/L) 1004.2 .+-. 2168.6 1344.8 .+-. 1780.8
1923.1 .+-. 2228.9 4195.2 .+-. 3721.5 <0.001 Male Sex 211 (85.8)
178 (72.3) 177 (72.0) 146 (59.6) <0.0001
TABLE-US-00002 TABLE 2 Multivariate Cox proportional hazards
regression model of significant predictors of death or heart
failure Variable Hazard Ratio 95% CI p value Log MR-proADM 3.63
1.48-8.90 0.005 Log NTproBNP 2.67 1.82-3.90 0.0001 Age 1.03
1.02-1.05 0.0001 Sex 0.69 0.46-0.96 0.031 PMH of AMI 1.76 1.24-2.50
0.001 Log creatinine 4.05 0.99-16.67 0.052
MR-proADM Levels in Patients
[0054] Plasma levels of MR-proADM in patients with AMI ranged from
0.09-6.66 nmol/L with a median of 0.73 nmol/L, being elevated
compared to the established normal range (mean 0.33, range
0.10-0.64 nmol/L)..sup.10 MR-proADM was significantly higher in
patients who died (1.31; [0.09-5.39] vs. 0.71; [0.25-6.66] nmol/L;
p<0.0001) or were readmitted with heart failure (1.10;
[0.40-4.39] vs. 0.71; [0.25-6.66] nmol/L; p<0.0001) compared to
event free survivors. There was a significantly higher level in
females compared with males (p<0.0001), in patients with a PMH
of AMI (p<0.0001), in those with a history of hypertension
(p<0.0001) and in patients who had a past history of heart
failure (p=0.001). MR-proADM levels were not significantly
different between STEMI and NSTEMI patients. MR-proADM was lower in
patients who were thrombolysed (p=0.043).
[0055] There was correlation of MR-proADM with age (r.sub.s=0.552,
p<0.0001), log creatinine (r.sub.s=0.404, p<0.0001), Killip
class (r.sub.s=0.314, p<0.0001), and NTproBNP (r.sub.s=0.519,
p<0.0001).
NTproBNP Levels in Patients
[0056] NTproBNP was significantly higher in patients who died
(5929.3; [104.3-16994.2] vs. 839.0; [0.30-28886.8] pmol/L;
p<0.0001) or were readmitted with heart failure (3932.9;
[2.4312933.0] vs. 839.0; [0.3-28886.8] pmol/L; p<0.0001).
Significant differences in NTproBNP levels were noted between males
and females (788.7; [0.3-28886.8] vs. 1632.6; [5.7-24016.0]
pmol/ml; p<0.0001) and those with a Killip class above 1 (631.0;
[0.3-24016.0] vs. 1583.4; [0.3-28886.8] pmol/ml; (p<0.0001) and
in patients with a PMH of heart failure (668.6; [5.70-28886.8] vs.
2415.9; [89.6-12933.0] pmol/ml; p=0.001) or AMI (844.4;
[0.3-28886.8] vs. 1332.3; [0.3-11259.0] pmol/ml; p=0.03).
Relationship Between MR-proADM and Echocardiographic Parameters
[0057] MR-proADM and NTproBNP were higher in patients with impaired
LV systolic function (as defined by EF<40% or LVWMI>1.8 on
echocardiography) (median [range] 0.91; [0.25-6.66] vs. 0.69;
[0.09-5.57]; nmol/L, p<0.0001) and 2286.1; [0.3-16994.2] vs.
804.6; [0.3-28886.8]; pmol/L, p<0.0001), respectively.
Primary Endpoints: MR-proADM and NTproBNP as Predictors of Death
and Heart Failure
[0058] MR-proADM was raised in patients with death or heart failure
compared to survivors (median [range] nmol/L, 1.19; [0.09-5.39] vs.
0.71; [0.25-6.66]; p<0.0001). When clinical and demographic
characteristics (age, sex, PMH of AMI, Killip class, log
creatinine, NTproBNP and MR-proADM), were entered into a
multivariate binary logistic model MR-proADM (OR 4.22, 95% CI:
1.25-14.26, p=0.02) and NTproBNP (OR 3.20, 95% CI: 2.07-4.94,
p<0.0001) independently predicted the primary endpoint along
with age (OR 1.04), gender (OR for male vs female 0.65), PMH of AMI
(OR 2.51) and log creatinine (OR 8.25). The Nagelkerke r.sup.2 was
0.35 suggesting a good fit of the model. Killip class was no longer
an independent predictor of death and heart failure. The
receiver-operating-characteristic curve for MR-proADM yielded an
area under the curve (AUC) of 0.77 (95% CI: 0.72-0.81, p<0.001);
for NTproBNP the AUC was 0.79 (95% CI: 0.75-0.84, p<0.001). The
predicted probability from the binary logistic model combining the
2 markers yielded an AUC of 0.84 (95% CI: 0.81-0.88, p<0.001),
which exceeded that of either peptide alone (FIG. 3).
[0059] Cox proportional hazards modelling confirmed that the same
variables (namely MR-proADM, NTproBNP, age, gender, PMH of AMI and
log creatinine) were independent predictors of death or heart
failure (Table 2).
[0060] The Kaplan-Meier survival curve revealed a significantly
better clinical outcome in patients with MR-proADM below the median
(0.73 nmol/L) compared with those with MR-proADM above the median
(log rank 61.27, p<0.0001, FIG. 4). This was also true for
NTproBNP (log rank 68.27, p<0.0001, FIG. 5). In patients
stratified by NTproBNP (median 914 pmol/L), MR-proADM gave
additional information on death and heart failure in those patients
who had NTproBNP level above the median (log rank for trend 57.22,
p<0.0001, FIG. 6), and even for patients below the NTproBNP
median value, MR-proADM had some predictive value (log rank for
trend 8.72, p=0.033, FIG. 6). Patients in the top quartile for
MR-proADM (above 1.04 nmol/L) had a significantly higher mortality
than those in quartiles 1 to 3 (p<0.0001 for all). For NTproBNP
below the median, those patients in the top quartile of MR-proADM
had higher, event rates than those in quartile 1 (p=0.006) and 2
(p=0.018), (FIG. 6).
[0061] The event rates at 1 year for both death and heart failure
readmission or death alone in patients stratified by median
NTproBNP (914 pmol/L) and quartiles of MR-proADM are illustrated in
FIG. 7, in which the top quartile of MR-proADM (1.04 nmol/L)
predicted those at highest risk.
Secondary Endpoints: MR-proADM and NTproBNP as Predictors of
Death
[0062] As described above both markers were significantly raised in
patients who died. On Cox proportional hazards modelling the
strongest independent predictors of death were MR-proADM (HR 4.86,
95% CI: 1.98-11.95, p=0.001) and NTproBNP (HR 3.64, 95%
CI:2.20-6.03, p<0.0001), the other independent predictors were
age (HR 1.06, p<0.0001), and PMH of AMI (HR 1.64, p=0.019).
Kaplan-Meier analysis revealed a significantly better clinical
outcome in patients with MR-proADM below the median compared with
those with MR-proADM above the median (log rank 42.40,
p<0.0001). In addition quartiles of MR-proADM predicted those
with the highest mortality, stratified by NTproBNP levels above the
median (log rank for trend 44.76, p<0.0001) and those in the top
MR-proADM quartile had higher mortality than those in quartiles 1-3
(p<0.0005, p<0.0001, p<0.0001 respectively).
Secondary Endpoints: MR-proADM and NTproBNP as Predictors of Heart
Failure
[0063] As described above both markers were significantly raised in
patients who were readmitted with heart failure.
[0064] On Cox proportional hazards modelling the independent
predictors of heart failure were MR-proADM (HR 7.29, 95% CI:
2.45-21.67, p<0.0001), NTproBNP (HR 1.71, 95% CI: 1.04-2.81,
p=0.034), Killip class above 1 (HR 2.04, 95% CI: 1.16-3.59,
p=0.014), and PMH of AMI (HR 1.93, 95% CI: 1.16-3.19, p=0.011).
[0065] The Kaplan-Meier survival curve revealed a significantly
better clinical outcome in patients with MR-proADM below the median
compared with those with MR-proADM above the median (log rank
28.65, p<0.0001). Following stratification by the median
NTproBNP level, quartiles of MR-proADM predicted those with the
highest readmission rate for heart failure especially in those with
NTproBNP above the median (log rank for trend 21.1, p<0.0001)
and those in MR-proADM quartiles 2 (p<0.027), 3 (p=0.0008) and 4
(p=0.002) had higher readmission rates for heart failure than those
in quartile 1.
Secondary Endpoints: MR-proADM and NTproBNP as Predictors of
Myocardial Infarction
[0066] Compared to survivors with no endpoints, patients who were
readmitted with AMI had similar NTproBNP (median [range] 890.4;
[0.3-28886] pmol/L vs. 1440.5; [2.6-10646.3]; p=NS) and MRproADM
levels (median [range] 0.73; [0.09-6.66] nmol/L vs. 0.75;
[0.31-2.00]; p=NS).
Discussion
[0067] This is the first report investigating the prognostic
potential of MR-proADM in a large cohort of patients from a single
centre and comparing this with NTproBNP, a well-established marker
of death and heart failure. Our data indicate by survival analysis
using both Kaplan-Meier and Cox proportional hazard models that
MR-proADM is a powerful independent predictor of death and heart
failure, with combined levels of MR-proADM and NTproBNP giving
independent prognostic information. Neither marker, however, was
predictive of recurrent myocardial infarction.
[0068] Reperfusion therapy and the application of secondary
prevention therapies have improved mortality post AMI. Despite
this, outcome remains poor for some patients..sup.22 Risk
stratification at an early stage after AMI remains important and
may be useful in helping to select treatment regimes or
investigation pathways in the future. A multimarker strategy for
outcome post-AMI using independent biomarkers has benefits in that
it integrates the different pathways involved in the hope that
complementary information can be gained..sup.23 Although from ROC
curve analysis, NTproBNP was the more accurate marker for
predicting outcome, MR-proADM levels provided complementary
prognostic information. The combination of MR-proADM and NTproBNP
in a multi-marker risk stratification approach generated an
increased area under the ROC curve and greater predictive accuracy.
Kaplan-Meier analysis revealed MR-proADM was particularly useful in
the in the group of patients who had a raised NTproBNP (above about
900 pmol/L). Levels of MR-proADM were predictive of poor outcome
especially in those with levels above the top quartile (1.04
nmol/L). Multivariate analyses (binary and the more statistically
powerful Cox regression) demonstrated that MR-proADM and NTproBNP
both retained statistically significant power for prediction of
death and heart failure independent of other demographic and
clinical variables. These findings have been obtained from a
heterogeneous AMI population with varying pre morbid cardiovascular
disease and variations in the treatments offered to patients
particularly with regards to thrombolysis.
[0069] The stimuli to the secretion of both markers may be similar;
this is backed up by significantly greater values of MR-proADM and
NTproBNP in patients with left ventricular systolic dysfunction. In
addition, there are similarities in MR-proADM and NTproBNP levels
between males and females, (both peptide levels higher in females
compared to males) and both levels increase with age from what has
been measured so far: Less pronounced for MR-proADM than for
NTproBNP.
[0070] NTproBNP is a more stable by-product in the production of
BNP..sup.24 In similar fashion MR-proADM is the more stable
by-product of ADM released in a 1:1 ratio. The benefit of measuring
the prohormones over the active peptide is that the lack of
receptor binding or protein interactions and the longer half-lives
result in higher plasma levels. The prohormones are also more
stable in blood ex-vivo, and this makes them generally more
applicable in clinical practice..sup.10
[0071] ADM may have a number of advantageous effects in the
post-AMI period, causing vasodilation (with reduction of arterial
and cardiac filling pressures) at a time when the myocardium has
been compromised and may cause increased myocardial contractility
via its downstream actions on cAMP..sup.6 ADM may also play a role
in maintaining sodium balance, inhibiting the production of
aldosterone despite an elevated renin activity, and thereby
optimizing cardiac filling at a time when the ventricle has taken
an insult..sup.25 ADM has been investigated previously in the post
AMI period where it was found to be weakly predictive of
death..sup.1 However its independent predictive power was lost for
death and heart failure when NTproBNP was evaluated. Interestingly,
it was not found to be raised in patients who later died or
developed heart failure.sup.1 but this may have been due to the
size of population investigated and possibly the fact that ADM has
a short half life, and is bound to receptors and other binding
proteins..sup.10 In another study ADM was found to be an
independent predictor of death and cardiogenic shock post
AMI..sup.17 ADM has also been shown to be raised in heart
failure.sup.16,26 with levels increasing with the severity of NYHA
class..sup.15 The current findings confirm that the ADM system may
be another candidate neurohormonal pathway, in addition to the
renin-angiotensin and sympathetic nervous systems that may be
associated with poor outcome after AMI. The confirmation of the
independent predictive value of MR-proADM together with NTproBNP
may have been achieved due to the design of the MR-proADM assay
which measures prohormone that does not bind to binding proteins or
receptors, with a resultant short half life..sup.10 This further
strengthens the concept of measurement of prohormones in disease
states, as plasma levels of these biologically inactive fragments
are generally higher than the active hormone.
CONCLUSION
[0072] Activation of the adrenomedullin system post AMI and
MR-proADM is a powerful new prognostic marker of death or heart
failure and the combined endpoint of both outcomes, in patients
with AMI, independent of established conventional risk factors and
newer plasma biomarkers such as NTproBNP. A multimarker approach
with MR-proADM and NTproBNP is more informative than either marker
alone, and may be useful for risk stratification in AMI patients,
with the possibility of changes in the investigation and therapy of
such individuals.
[0073] All of the references described or referred to herein are
incorporated by reference in their entirety for all useful
purposes.
[0074] While certain specific structures embodying the invention
are shown and described, it will be obvious to those skilled in the
art that various modifications and rearrangements of the parts may
be made without departing from the spirit and scope of the
underlying inventive concept and that said invention is not limited
to the particular forms shown and described herein.
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Sequence CWU 1
1
21185PRTHomo sapiens 1Met Lys Leu Val Ser Val Ala Leu Met Tyr Leu
Gly Ser Leu Ala Phe 1 5 10 15 Leu Gly Ala Asp Thr Ala Arg Leu Asp
Val Ala Ser Glu Phe Arg Lys 20 25 30 Lys Trp Asn Lys Trp Ala Leu
Ser Arg Gly Lys Arg Glu Leu Arg Met 35 40 45 Ser Ser Ser Tyr Pro
Thr Gly Leu Ala Asp Val Lys Ala Gly Pro Ala 50 55 60 Gln Thr Leu
Ile Arg Pro Gln Asp Met Lys Gly Ala Ser Arg Ser Pro 65 70 75 80 Glu
Asp Ser Ser Pro Asp Ala Ala Arg Ile Arg Val Lys Arg Tyr Arg 85 90
95 Gln Ser Met Asn Asn Phe Gln Gly Leu Arg Ser Phe Gly Cys Arg Phe
100 105 110 Gly Thr Cys Thr Val Gln Lys Leu Ala His Gln Ile Tyr Gln
Phe Thr 115 120 125 Asp Lys Asp Lys Asp Asn Val Ala Pro Arg Ser Lys
Ile Ser Pro Gln 130 135 140 Gly Tyr Gly Arg Arg Arg Arg Arg Ser Leu
Pro Glu Ala Gly Pro Gly 145 150 155 160 Arg Thr Leu Val Ser Ser Lys
Pro Gln Ala His Gly Ala Pro Ala Pro 165 170 175 Pro Ser Gly Ser Ala
Pro His Phe Leu 180 185 248PRTHomo sapiens 2Glu Leu Arg Met Ser Ser
Ser Tyr Pro Thr Gly Leu Ala Asp Val Lys 1 5 10 15 Ala Gly Pro Ala
Gln Thr Leu Ile Arg Pro Gln Asp Met Lys Gly Ala 20 25 30 Ser Arg
Ser Pro Glu Asp Ser Ser Pro Asp Ala Ala Arg Ile Arg Val 35 40
45
* * * * *
References