U.S. patent application number 15/017140 was filed with the patent office on 2016-06-09 for diagnosis and risk stratification using nt-proet-1.
This patent application is currently assigned to Brahms GmbH. The applicant listed for this patent is Brahms GmbH. Invention is credited to Andreas Bergmann, Joachim Struck.
Application Number | 20160161505 15/017140 |
Document ID | / |
Family ID | 39745333 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160161505 |
Kind Code |
A1 |
Bergmann; Andreas ; et
al. |
June 9, 2016 |
DIAGNOSIS AND RISK STRATIFICATION USING NT-proET-1
Abstract
The invention relates to a method for the diagnosis and/or risk
stratification of cardiac diseases and diseases of the respiratory
tract and lungs. According to said method, the free fragment
N-terminal proEndothelin (NT-proET-1; amino acids 18-52 of the
preproET according to FIG. 1) or fragments and partial peptides
thereof is or are determined.
Inventors: |
Bergmann; Andreas; (Berlin,
DE) ; Struck; Joachim; (Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Brahms GmbH |
Hennigsdorf |
|
DE |
|
|
Assignee: |
Brahms GmbH
Hennigsdorf
DE
|
Family ID: |
39745333 |
Appl. No.: |
15/017140 |
Filed: |
February 5, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12599197 |
Jan 12, 2010 |
9261516 |
|
|
PCT/DE2008/000781 |
May 8, 2008 |
|
|
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15017140 |
|
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Current U.S.
Class: |
436/501 ;
422/69 |
Current CPC
Class: |
G01N 2333/5754 20130101;
G01N 2800/32 20130101; G01N 2800/122 20130101; G01N 2800/52
20130101; G01N 33/74 20130101; G01N 2800/12 20130101; G01N 2800/50
20130101; G01N 33/6893 20130101; G01N 2800/324 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 8, 2007 |
DE |
10 2007 021 443.1 |
Claims
1. A method for the in-vitro diagnosis and/or risk stratification
of heart diseases and/or diseases of the lungs and respiratory
tract, determining the free fragment NT-proET-1 (18-52), or
fragments and partial peptides thereof, in a patient to be
examined.
2. The method for the diagnosis and/or risk stratification of heart
diseases according to claim 1, characterized in that the heart
diseases are selected from acute coronary syndrome, post-myocardial
infarction, and cardiac insufficiency.
3. The method for the diagnosis and/or risk stratification of
diseases of the lungs and respiratory tract according to claim 1,
characterized in that they are selected from infections cause by
bacteria, viruses, fungi or parasites, particularly lower
respiratory tract infections (LRTI), bronchitis, pneumonia,
sarcoidosis, bronchiectases, noncardiac pulmonary edema and/or
chronic diseases of the lungs and respiratory tract, particularly
interstitial lung diseases and pulmonary fibrosis, chronic
obstructive pulmonary disease (COPD), particularly COPD infection
exacerbations, bronchial asthma, particularly infection
exacerbations with bronchial asthma.
4. A method for the diagnosis and/or risk stratification according
to claim 1, for the identification of patients that are at
increased risk and/or have an unfavorable prognosis for heart
diseases and/or diseases of the lungs and respiratory tract.
5. A method for the diagnosis and/or risk stratification according
claim 1 , wherein the patient is a symptomatic and/or asymptomatic
patient, particularly an emergency patient.
6. A method for the diagnosis and/or risk stratification according
to claim 1 , for the control of the therapy of heart diseases
and/or diseases of the lungs and respiratory tract, particularly in
intensive care or emergency care.
7. A method for the diagnosis and/or risk stratification of heart
diseases and/or diseases of the lungs and respiratory tract
according to claim 1, for the execution of clinical decisions,
particularly advanced treatments and therapies using drugs,
particularly in intensive care or emergency care, to include the
decision to hospitalize the patient.
8. A method for the diagnosis and/or risk stratification of heart
diseases and/or diseases of the lungs and respiratory tract
according to claim 1, for the prognosis, early detection and
detection by differential diagnosis, assessment of the severity,
and assessment of the course of the disease concomitant with the
therapy.
9. A method according to claim 1, characterized in that
additionally a determination of at least one further marker is
carried out, selected from the group of inflammatory markers,
cardiovascular markers, neurohormonal markers, or ischemic
markers.
10. A method according to claim 9, characterized in that the
inflammatory marker is selected from at least one marker of the
group of C-reactive protein (CRP), cytokines such as TNF-alpha,
interleukines such as IL-6, procalcitonin (1-116, 3-116), and
adhesion molecules such as VCAM or ICAM.
11. A method according to claim 9, characterized in that the
cardiovascular marker is selected from at least one marker of the
group creatine kinase, myoglobin, myeloperoxidase, natriuretic
protein, particularly ANP (or ANF), proANP, NT-proANP, BNP, proBNP,
NT-proBNP, or a partial sequence thereof, cardiac troponin, CRP,
and also regulatory (pro)hormones, such as pro-gastrin releasing
peptide (pro-GRP), pro-endothelin-2, pro-endothelin-3, pro-leptin,
pro-neuropeptide Y, pro-somatostatin, pro-neuropeptide YY,
pro-opionmelanocortin, pro-adrenomedullin (pro-ADM),
pro-vasopressin (pro-AVP), or a partial sequence thereof.
12. A method according claim 9, characterized in that the ischemic
marker is selected from at least one marker of the group troponin I
and T, CK-MK.
13. A method according to claim 9, characterized in that the
neurohormonal marker is at least one natriuretic protein,
particularly ANP (or ANF), proANP, NT-proANP, BNP, proBNP,
NT-proBNP, or a partial sequence thereof.
14. A method according to claim 9, characterized in that parallel
or simultaneous determinations of the markers are carried out.
15. A method according to claim 1, characterized in that the
determinations are carried out using at least one patient
sample
16. A method according to claim 1, characterized in that the
determinations are carried out using an automatic analysis system,
particularly by means of a Kryptor.
17. A method according to claim 1, characterized in that the
determinations are carried out using a rapid test, particularly in
individual or multiple parameter determinations.
18. Use of the free fragment NT-proET-1, or fragments and partial
peptides thereof, for the diagnosis and/or risk stratification of
heart diseases and/or diseases of the lungs and respiratory tract,
and optionally further markers, according to claim 9.
19. A kit for the diagnosis and/or risk stratification of heart
diseases and/or diseases of the lungs and respiratory tract,
comprising detection reagents for the determination of the free
fragment NT-proET-1, or fragments and partial peptides thereof, and
optionally further markers, according to claim 9 and auxiliary
agents.
20. A diagnostic device for carrying out a method according to
claim 1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/599,197, filed Jan. 12, 2010, which is a national stage
application (under 35 U.S.C. .sctn.371) of PCT/DE2008/000781, filed
May 8, 2008, which claims benefit of German application
102007021443.1, filed May 8, 2007. The entire contents of each of
these applications are hereby incorporated by reference herein.
SUBMISSION OF SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is
14519_68_Sequence_Listing. The size of the text file is 4 KB, and
the text file was created on Feb. 5, 2016.
DESCRIPTION
[0003] The invention relates to a method for the diagnosis and/or
risk stratification of heart diseases and diseases of the
respiratory tract and lungs, wherein a determination of the free
fragment N-terminal proendothelin (NT-proET-1; AS 18-52 of the
preproET according to FIG. 1), or fragments and partial peptides
thereof, is carried out.
[0004] In order to apply a suitable therapy, an early diagnosis and
differentiation of heart diseases and/or diseases of the
respiratory tract and lungs are necessary already in the emergency
room, in conjunction with the need to make clinical decisions. In
the state of the art, mature ET-1 (21 AS) is described for
diagnostics purposes, including the precursor protein Big-ET1
(38AS, refer to FIG. 1) (Rossi G P, Seccia T. M., Albertin G,
Pessina A C Measurement of endothelin: clinical and research use.
Ann Clin Biochem 2000; 37(Pt 5) : 608-26.2; Aubin P, Le Brun G,
Moldovan F, Villette J M, Creminon C, Dumas J, et al. Sandwich-type
enzyme immunoassay for big endothelin-I in plasma: concentrations
in healthy human subjects unaffected by sex or posture. Clin Chem
1997; 43: 64-70). Furthermore, it is known that endothelin
prohormones for the diagnosis of sepsis are disclosed in WO
00/22439 and EP 1121600 B1 by the applicant.
[0005] In addition, the C-terminal proendothelin fragments
(CT-proET-1) having the amino acid sequences 93-212 or 168-212 of
the preproendothelin (FIG. 1, SEQ ID No. 1) for the diagnosis of
cardiovascular diseases for the indirect determination of the
endothelin-1 or the big-endothelin-1 content are described in EP
1564558 B1.
[0006] The disadvantage of the known diagnostic methods, using the
markers known so far, however, continues to be that an early and
comprehensive detection of risk patients is not always sufficiently
successful, and risk stratification therefore is not sufficient. An
object of the invention is therefore to develop a method for the
risk stratification of heart diseases and diseases of the
respiratory tract and lungs that enables improved detection of risk
patients.
[0007] Furthermore it is disadvantageous that in the prior art
typically the sensitivity and/or specificity of the markers is
insufficient.
[0008] Another object is therefore to provide a method for the risk
stratification of heart diseases and diseases of the respiratory
tracts and lungs, wherein at least one marker, or a combination of
markers, has sufficient sensitivity and specificity in an in-vitro
diagnosis.
[0009] It is therefore the object of the present invention to
provide a method for the diagnosis and/or risk stratification of
heart diseases and/or diseases of the respiratory tracts and
lungs.
[0010] The object is achieved by a method for the diagnosis and
risk stratification of heart diseases and diseases of the
respiratory tract and lungs, wherein a determination of the
N-terminal proendothelin (in short: "NT-proET-1"), or fragments and
partial peptides thereof, is carried out (hereafter the method
according to the invention).
[0011] Surprisingly, NT-proET-1, or fragments and partial peptides
thereof, exhibit a high sensitivity and specificity for the
diagnosis of heart diseases and diseases of the respiratory tract
and lungs (refer to examples and figures).
[0012] The term "heart diseases" according to the invention in
particular includes the "acute coronary syndrome" and the different
phases thereof of coronary heart disease, which are immediately
life-threatening. This applies in particular to emergency medicine,
and specifically to acute myocardial infarction and/or angina
pectoris and also sudden cardiac death. In addition to acute
myocardial infarction, which according to WHO criteria (WHO (1979):
Nomenclature and criteria for diagnosis of ischemic heart disease.
Report of the Joint International Society and Federation of
Cardiology/World Health Organization task force on standardization
of clinical nomenclature, Circulation 59 (3) : 607-609) is defined
as an acute chest pain event lasting for more than 20 minutes,
associated with changes in the ST segment elevation, and/or an
increase in myocardial enzymes, the term unstable angina pectoris
(AP) was coined, which according to the invention is to be
considered part of "heart diseases" (Hamm C W: Leitlinien: Akutes
Koronarsyndrom (ACS)-Teil 1: ACS ohne persistierende S-T-Hebung
[Guidelines: Acute Coronary Syndrome (ACS)--Part 1: ACS without
persistent ST segment elevation]. Z Kardiol (2004) 93:72-90).
[0013] As part of the present invention, "myocardial infarction"
(heart attack, AMI (acute myocardial infarction)) shall denote an
acute and life-threatening disease of the heart, wherein damage or
death (infarction) of parts of the heart muscle (myocardium) occurs
due to a restriction in blood supply (ischemia), which typically is
present for more than 20 minutes. The cardinal symptom of a heart
attack is suddenly occurring chest pain, which lasts for more than
20 minutes and is usually severe, and which can radiate into the
shoulders, arms, lower jaw and upper abdomen and may be accompanied
by sweating, nausea and possibly vomiting. As a result of
myocardial infarction, heart failure is possible.
[0014] The term "post-myocardial infarction" means that a patient
suffered from myocardial infarction in the past, which is to say,
for example, more than 1 hour, in particular 20 hours, above all 1
to 5 days, or 3 to 5 days earlier, and is now living in the post
phase, wherein the incident did not result in immediate death, but
directly and indirectly a further disadvantageous event is to be
expected, such as another (subsequent) myocardial infarction, heart
failure, or death, or otherwise deterioration of the patient.
[0015] Within the context of the present invention, "cardiac
insufficiency" shall also be included in "heart diseases".
[0016] "Cardiac insufficiency" is an acute or chronic inability of
the heart to supply enough blood, and consequently sufficient
oxygen, to the tissue in order to ensure tissue metabolism at rest
or during physical activity. Clinically, cardiac insufficiency
exists when typical symptoms (dyspnea, fatigue, fluid retention)
are present, the underlying cause of which is a functional cardiac
disorder in the sense of systolic or diastolic dysfunction. Chronic
cardiac insufficiency (CCI) is also included according to the
invention (Kardiologie compact, published by Chrisian Mewis, Reimer
Riessen und loakim Spyridopoulos, 2nd unrevised edition, Thieme
2006).
[0017] The following can be causes of cardiac insufficiency:
valvular heart defect (such as a long-term consequence of rheumatic
fever), myocarditis (inflammation of the heart muscle), cardiac
dysrhythmias, cardiac infarction, in addition to high blood
pressure (hypertension) and/or arteriosclerosis (calcification) of
the coronary vessels (coronary heart disease). Also included
according to the invention are hypertensive heart diseases with
(congestive) heart failure, hypertensive heart and kidney disease
with (congestive) heart failure, primary right heart failure,
secondary right heart failure, left heart failure without
discomfort (NYHA stage I), left heart failure with discomfort
during higher physical activity (NYHA stage II), left heart failure
with discomfort during light physical activity (NYHA stage III),
left heart failure with discomfort at rest (NYHA stage IV), and
cardiogenic shock.
[0018] Within the context of the present invention, "diseases of
the respiratory tract and lungs" shall mean in particular
"infections of the lungs and respiratory tract", specifically such
infections caused by bacteria, viruses, fungi, or parasites, such
as indications like lower respiratory tract infections (LRTI),
bronchitis, pneumonia, sarcoidosis, bronchiectases, or noncardiac
pulmonary edema. Additionally, according to the invention lower
respiratory tract infections (LRTI), bronchitis, putrid bronchitis,
and pneumonia are preferred. Particularly preferred is pneumonia,
in particular community-associated pneumonia (CAP), and lower
respiratory tract infections (LRTI).
[0019] Within the context of this invention, pneumonia shall be
interpreted as an acute or chronic disease of the pulmonary tissue,
the infection of which is caused by bacteria, viruses or fungi,
parasites, rarely also toxically by the inhalation of toxic
substances, or immunologically. For the clinician, pneumonia is a
combination of different symptoms (fever or hypothermia, chills,
coughing, pleuritic pain of the thorax, increased sputum
production, increased respiratory rate, percussion sound damping,
bronchial respiration, high-pitched rales, pleural friction sound),
in conjunction with at least one infiltrate recognizable on the
thorax x-ray image (Harrisons Innere Medizin [Harrison's Internal
Medicine], published by Manfred Dietel, Norbert Suttorp and Martin
Zeitz, A B W Wissenschaftsverlage 2005).
[0020] Within the context of this invention, "diseases of the
respiratory tract and lungs" in particular denotes "chronic
diseases of the lungs and respiratory tract", specifically such
indications like interstitial lung disease and pulmonary fibrosis,
chronic obstructive pulmonary disease (COPD), particularly COPD
infection exacerbations, bronchial asthma, particularly infection
exacerbations with bronchial asthma, and bronchial carcinoma.
Particularly preferred is COPD, in particular COPD infection
exacerbations.
[0021] According to the invention, COPD denotes a group of chronic
diseases, which are characterized by coughing, increased
expectoration, and shortness of breath during physical activity.
Primarily, chronic-obstructive bronchitis and pulmonary emphysema
should be mentioned. Both disease patterns are characterized in
that especially expiration is labored. A colloquial term for the
primary symptom of COPD is also "smoker's cough". The invention is
particularly advantageous for acute exacerbations.
[0022] All indications mentioned above are also described, for
example, in Pschyrembel, De Gruyter, 9th edition, Berlin 2004.
[0023] According to the invention, the term "risk stratification"
comprises the identification of patients, particularly emergency
patients and risk patients, having poor prognoses, for the purpose
of more intensive diagnostics and therapy/treatment of heart
diseases, particularly the acute coronary syndrome and myocardial
infarction, angina pectoris and/or post-myocardial infarction,
cardiac insufficiency, or diseases of the respiratory tract and
lungs, particularly infections and chronic inflammations, with the
goal of enabling the most favorable course of the disease possible.
Risk stratification according to the invention consequently allows
an effective treatment method, which exists for example in the case
of the acute coronary syndrome in the form of percutaneous coronary
interventions and newer drugs, or for the treatment or therapy of
cardiac insufficiency, for example, using ACE inhibitors, AT1
antagonists, angiotensin II receptor blockers (subtype 1), the beta
blockers bisoprolol, carvedilol, metoprolol and nebivolol,
vasopressin receptor antagonists, aldosterone antagonists starting
with NYHA stage Ill, calcium sensitizers (levosimendan), or for the
treatment or therapy of infections or chronic diseases of the
respiratory tract and lungs using antibiotics.
[0024] The invention therefore likewise relates to the
identification of patients that are at increased risk and/or have
an unfavorable prognosis for heart diseases and diseases of the
respiratory tract and lungs, specifically symptomatic and/or
asymptomatic patients, particularly emergency patients.
[0025] Particularly in cases in emergency and/or intensive care,
reliable stratification can be carried out particularly
advantageously by using the method according to the invention. The
method according to the invention thus enables clinical decisions
that result in fast success of the therapy and the prevention of
fatalities. Such clinical decisions also comprise the advanced
treatment with drugs for the treatment or therapy of heart diseases
and diseases of the respiratory tract and the lungs.
[0026] The invention therefore also relates to a method for the
diagnosis and/or risk stratification of patients with heart
diseases and/or diseases of the respiratory tract and lungs for the
execution of clinical decisions, such as advanced treatment and
therapy using drugs, preferably in time-critical intensive or
emergency care, to include the decision to hospitalize the
patient.
[0027] In a further preferred embodiment, the method according to
the invention therefore relates to the control of the therapy for
heart diseases and/or diseases of the respiratory tract.
[0028] A further preferred embodiment, the method according to the
invention for the diagnosis and/or risk stratification [is carried
out] for the prognosis, early detection and detection by
differential diagnosis, assessment of the severity, and assessment
of the course of the disease concomitant with therapy.
[0029] In a further preferred embodiment, the invention relates to
a method for in-vitro diagnostics for the early or differential
diagnosis or prognosis of heart diseases and/or diseases of the
respiratory tract and lungs, wherein a determination of the
NT-proET-1 marker, or fragments and partial peptides thereof, is
carried out in a patient to be examined.
[0030] In one embodiment of the method according to the invention,
body fluid, preferably blood, optionally whole blood or serum or
available plasma, is sampled from the patient to be examined, and
the diagnosis is made in vitro/ex vivo, which is to say outside of
the human or animal body. Due to the determination of the
NT-proET-1 marker, or fragments and partial peptides thereof, a
high sensitivity and specificity are achieved (refer to examples
and figures), and the diagnosis or risk stratification is possible
based on the available quantity in at least one patient sample.
[0031] Within the context of the present invention, "NT-proET-1"
shall be interpreted as a human protein or polypeptide, which can
be obtained from preproendothelin and, within the framework of
preproendothelin (SEQ ID No. 1 and FIG. 1) may comprise the free
fragments having amino acids 18-52 and fragments or partial
peptides that can be obtained therefrom, particularly
APETAVLGAELSAV (SEQ ID No. 2) Pos. 18-31 of preproET-1 (SEQ ID No.1
and FIG. 1) and GENGGEKPTPSPPWRLRRSKR (SEQ ID No. 3) Pos. 32-52 of
preproET-1 (SEQ ID No. 1 and FIG. 1). Furthermore, these
polypeptides according to the invention may have posttranslational
modifications, such as glycolization [sic], lipid attachment or
derivatization.
[0032] In a further embodiment, the determination of NT-proET-1, or
fragments and partial peptides thereof, can additionally be carried
out with further markers, specifically preferably such which
already point to a heart disease, particularly coronary heart
disease, and/or diseases of the respiratory tract and lungs.
[0033] As a result, the invention relates to such an embodiment of
the method according to the invention, wherein the determination is
additionally carried out in a patient to be examined using at least
one further marker selected from the group of inflammatory markers,
cardiovascular markers, neurohormonal markers, or ischemic
markers.
[0034] According to the invention, the inflammatory marker may be
selected from at least one marker of the group of C-reactive
protein (CRP), cytokines such as TNF-alpha, interleukins such as
IL-6, procalcitonin (1-116, 3-116), and adhesion molecules such as
VCAM or ICAM, and the cardiovascular marker may in particular be a
marker indicating necrosis of the tissue of the heart muscle, and
the marker influencing blood pressure can be selected from at least
one marker of the group of creatine kinase, myoglobin,
myeloperoxidase, natriuretic protein, particularly ANP (or ANF),
proANP, NT-proANP, BNP, proBNP, NT-proBNP, or a partial sequence
thereof, cardiac troponin, CRP. Furthermore, it shall also include
regulatory (pro)hormones, particularly pro-gastrin releasing
peptide (pro-GRP), proendothelin-2, pro-endothelin-3, pro-leptin,
pro-neuropeptide Y, pro-somatostatin pro-neuropeptide YY,
pro-opionmelanocortin, pro-adrenomedullin (pro-ADM),
pro-vasopressin (pro-AVP), or a partial sequence thereof. The
ischemic marker may be selected from at least one marker of the
group troponin I and T, CK-MB. In addition, the neurohormonal
marker can be at least one further natriuretic protein,
particularly ANP (or ANF), proANP, NT-proANP, BNP, proBNP, or a
partial sequence thereof.
[0035] In a further embodiment of the invention, the method
according to the invention can be carried out as part of an
in-vitro diagnosis using parallel or simultaneous determinations of
the markers (for example, multititer plates having 96 and more
wells), wherein the determinations are carried out using at least
one patient sample.
[0036] Furthermore, the method according to the invention and the
determinations thereof can be carried out in a diagnostic device
based on an automatic analysis system, particularly by means of a
Kryptor (http://www.kryptor.net/).
[0037] In a further embodiment, the method according to the
invention and the determinations thereof can be carried out using a
rapid test (such as lateral flow test or Point of Care), be it in
an individual or multiple parameter determination. In a
particularly preferred embodiment, it is a self-test or a device
suitable for emergency diagnostics.
[0038] The invention further relates to the use of NT-proET-1, or
fragments and partial peptides thereof, for the risk stratification
of heart diseases and/or diseases of the respiratory tract and
lungs and/or for in-vitro diagnostics for the early or differential
diagnosis or prognosis of heart diseases and/or diseases of the
respiratory tract and lungs.
[0039] Another object is to provide a corresponding diagnostic
device or the use thereof for carrying out the method according to
the invention.
[0040] Within the context of the present invention, such a
diagnostic device shall be interpreted in particular as an array or
assay (such as an immunoassay, ELISA and the like), in the broadest
sense a device for carrying out the method according to the
invention.
[0041] The invention further relates to a kit for the diagnosis
and/or risk stratification of heart diseases and/or diseases of the
respiratory tract and lungs, comprising detection reagents for the
determination of NT-proET-1, or fragments and partial peptides
thereof, optionally above-mentioned further markers. Such detection
reagents comprise antibodies and the like, for example.
[0042] The following examples and figures are provided for a more
detailed description of the invention, however without limiting the
invention to these examples and figures.
EXAMPLES AND FIGURES
[0043] Immunoassay
[0044] A sandwich immunoassay was used for measuring NT-proET-1 as
described in Struck et al (Struck J, Morgenthaler N. G., Bergmann
A. Proteolytic processing pattern of the endothelin-1 precursor in
vivo. Peptides 2005; 26;2482-6).
[0045] Peptide Synthesis:
[0046] Derived from the known amino acid sequence of preproET-1
(FIG. 1), two areas were selected (Pos. 18-31, 32-52). The areas
were chemically synthesized as soluble peptides, in one case
supplemented by an N-terminal cysteine residue, using standard
methods, and purified, the quality thereof was controlled using
mass spectrometry and reversed-phase HPLC, and they were
lyophilized in aliquots (JPT company, Berlin, Germany). The amino
acid sequences of the peptides are:
[0047] PAV15 CAPETAVLGAELSAV POS. 18-31 of preproET-1 (FIG. 1),
MPGC22 GENGGEKPTPSPPWRLRRSKRC POS. 32-52 of preproET-1 (FIG. 1) .
Furthermore, a peptide comprising the area Pos. 18-52 of preproET-1
was synthesized (PAR35 APETAVLGAELSAVGENGGEKPTPSPPWRLRRSKR).
[0048] Conjugation and Immunization
[0049] By means of MBS (m-maleimidobenzoyl-N-hydroxysuccinimide
ester), the peptides PAV15 and MPGC22 were conjugated to the KLH
(keyhole limpet hemocyanin) carrier protein (see instruction sheet
"NHS-Esters-Maleimide Crosslinkers" from PIERCE, Rockford, Ill.,
USA). These conjugates were used to immunize sheep according to the
following regimen: Each sheep initially received 100 .mu.g
conjugate (quantity information relative to the peptide portion of
the conjugate) and subsequently 50 .mu.g conjugate (quantity
information relative to the peptide portion of the conjugate) each
in 4 week intervals. Beginning with month four after start of the
immunization, 700 ml blood was collected from each sheep in 4 week
intervals and from it antiserum was obtained by centrifugation. The
conjugations, immunization, and production of antisera were carried
out by MicroPharm, Carmarthenshire, UK.
[0050] Purification of the Antibodies
[0051] In a one-step method, the peptide-specific antibodies were
produced from the antisera, which had been produced beginning with
month four after immunization. For this purpose, the peptides PAV15
and MPGC22 were first coupled to SulfoLink gel (refer to
instruction sheet "SulfoLink Kit" from PIERCE, Rockford, Ill.,
USA). For coupling, 5 mg peptide was provided per 5 ml of gel.
[0052] The affinity purification of peptide-specific antibodies
from sheep antisera against the peptides was carried out as
follows:
[0053] The peptide columns were first washed three times
alternately using 10 ml elution buffer (50 mM citric acid, pH 2.2)
and binding buffer (100 mN sodium phosphate, 0.1% Tween, pH 6.8).
100 ml of the antisera was filtered over 0.2 .mu.m and mixed with
the available column material. To this end, the gel was
quantitatively rinsed out of the column with 10 ml binding puffer.
The incubation was carried over night at room temperature under
pivoting motion. The batches were quantitatively transferred into
empty columns (NAP 25, Pharmacia, emptied). The passes were
discarded. Subsequently, a washing step was carried out with 250 ml
binding buffer without protein (protein content of the wash eluate
<0.02 A280 nm). Elution buffer was added to the washed columns,
and fractions of 1 ml each were collected. For each fraction, the
protein content was determined using the BCA method (refer to
instruction sheet from PIERCE, Rockford, Ill., USA). Fractions
having protein concentrations of >0.8 mg/ml were pooled. After
the protein determination of the pools using the BCA method, yields
of 27 mg were obtained for the anti-PAV15 antibody and 35 mg for
the anti-MPGC22 antibody.
[0054] Labeling
[0055] Using NAP-5 gel filtration columns (Pharmacia), 500 .mu.l of
the purified anti-MPGC22 antibody (see above) underwent a buffer
exchange in 1 ml 100 mM potassium phosphate buffer (pH 8.0)
according to the instruction sheet. The protein concentrations of
the antibody solution were adjusted to 1.5 mg/ml using 100 mM
potassium phosphate buffer (pH 8.0).
[0056] For chemiluminescence labeling, the antibody was further
treated as follows: 67 .mu.l of the antibody solution was mixed
with 10 .mu.l MA70-Akridinium-NHH-ester (1 mg/ml; HOECHST Behring)
and incubated at room temperature for 15 minutes. Then 423 .mu.l 1
M glycine was added and incubated for another 10 minutes.
[0057] Subsequently, the labeling batch underwent a buffer exchange
in 1 ml eluant A (50 mM potassium phosphate, 100 mM NaCl, pH 7.4)
using an NAP-5 gel filtration column (Pharmacia) in accordance with
the instruction sheet and in the process was freed of low-molecular
components. In order to separate the remaining labels not bound to
antibodies, gel filtration HPLC was carried out (column: Waters
Protein Pak SW 300). The sample was applied and chromatographed at
a flow rate of 1 ml/ml using eluant A. Using a photometer, the
wavelengths 280 nm and 368 nm were measured. The absorption ratio
of 368 nm/280 nm as a measure of the degree of labeling of the
antibody was 0.10+/-0.01 at the peak. The monomer
antibody-containing fractions (retention time 8-10 min) were
collected and placed in 3 ml 100 mM sodium phosphate, 150 mM NaCl,
5% bovine serum albumin, 0.1% sodium azide, pH 7.4.
[0058] Coupling
[0059] Irradiated 5 ml polystyrene tubes (Greiner) was coated as
follows with purified anti-PAV15 antibody: The antibody was diluted
in 50 mM Tris, 100 mM NaCl, pH 7.8 to a concentration of 6.6
.mu.g/ml. 300 of this solution was pipetted into each tube. The
tubes were incubated for 20 hours at 22.degree. C. The solution was
suctioned. Then, each tube was filled with 4.2 ml 10 mM sodium
phosphate, 2% Karion FP, 0.3% bovine serum albumin, pH 6.5. After
20 hours, the solution was suctioned. Finally, the tubes were dried
in a vacuum dryer.
[0060] Execution and Evaluation of the Immunoassay
[0061] The standard material used was peptide PAR35, which was
diluted serially in normal horse serum (SIGMA). The standards
produced in this way were allocated to concentrations in accordance
with the initial peptide weight.
[0062] The sandwich immunoassay was started as follows: 50 .mu.l
standards or samples and 200) .mu.l assay buffer (100 mM sodium
phosphate, 150 mM NaCl, 5% bovine serum albumin, 0.1% unspecific
sheep IgG, 0.1% sodium azide, pH 7.4), comprising 1 million RLU
(relative light units) of the MA70-labeled antibody, were pipetted
in the respective test tubes coated with antibodies. The incubation
was conducted for 2 hours at 22.degree. C. while shaking. Then, 4
washing steps were carried out using 1 ml washing solution (0.1%
Tween 20) each per tube, the fluid was allowed to drip off, and the
chemiluminescence bound to the tube was measured using a
luminometer (BERTHOLD, LB952T; base reagents BRAHMS AG). Using the
MultiCalc (Spline Fit) software, the NT-proET-1 concentrations of
the samples were read from the standard curve.
[0063] Analyte that can be measured using the described assay is
referred to as N-terminal proendothelin-1 (NT-proET-1).
[0064] Clinical Valency
[0065] Normal Range
[0066] NT-proET-1 concentrations were determined in samples of
healthy control persons (n=200). The median was 30.5 pmol/l, the
smallest value measured was 2.0, the highest one was 53 pmol/l, the
95th percentiles were 16.3 and 47.2 pmol/l.
[0067] Cardiac Insufficiency/Severity
[0068] NT-proET-1 concentrations were measured for patients with
chronic or acute decompensated cardiac insufficiency. NT-proET-1
concentrations were associated with the severity of the cardiac
insufficiency, particularly with increased severity: The medians of
NT-proET-1 concentrations for the four severity categories NYHA
I-IV were: 35.4, 37.3, 45.5 and 83.9 pmol/L (refer to FIG. 2).
[0069] Chronic Cardiac Insufficiency/Diagnosis
[0070] NT-proET-1 values were determined from a pool of 316
patients suffering from chronic cardiac insufficiency and 200
healthy control patients. The receiver-operating characteristic
analysis showed an AUC of 0.72. For a cut-off value of 49.7 pmol/l,
this produced a sensitivity of 34.3% with a specificity of 98%. For
a cut-off value of 47.2 pmol/l, this produced a sensitivity of
38.5% with a specificity of 95%.
[0071] Chronic Cardiac Insufficiency/pPrognosis
[0072] NT-proET-1 values were determined from a pool of 316
patients suffering from chronic cardiac insufficiency. The patients
were monitored for a mean period of 360 days. During this period,
42 patients passed away, 274 survived. Using the receiver-operating
characteristic analysis, the best cut-off value (defined as the
largest product of sensitivity and specificity) was determined for
the prognosis of the mortality rate: 51.3 pmol/l. At this cut-off
value, the sensitivity of the prognosis was 71.4%, the specificity
was 74.5%. The likelihood ratio of passing away at a cut-off value
of 51.3 pmol/l was 2.8.
TABLE-US-00001 TABLE 1 <51.3 pmol/l >51.3 pmol/l Survivors
204 70 Fatalities 12 30
[0073] Acute Cardiac Insufficiency/Diagnosis
[0074] NT-proET-1 values were determined from a pool of 125
patients suffering from acute shortness of breath. 69 of the 125
patients suffered from cardiac insufficiency. The receiver
operating characteristic analysis for the differential diagnosis of
cardiac insufficiency showed an AUC of 0.72. For a cut-off value of
111 pmol/l, this produced a sensitivity of 10.2% with a specificity
of 98%. For a cut-off value of 90.5 pmol/l, this produced a
sensitivity of 17.5% with a specificity of 95%.
[0075] Acute Cardiac Insufficiency/Prognosis
[0076] NT-proET-1 values were determined from a pool of 69 patients
suffering from acute decompensated cardiac insufficiency. The
patients were monitored for a period of 360 days. During this
period, 21 patients passed away, 48 survived. Using the
receiver-operating characteristic analysis, the best cut-off value
(defined as the largest product of sensitivity and specificity) was
determined for the prognosis of the mortality rate: 64 pmol/l. At
this cut-off value, the sensitivity of the prognosis was 66.6%, the
specificity was 68.7%. The likelihood ratio of passing away at a
cut-off value of 64 pmol/l was 2.0.
TABLE-US-00002 <64 pmol/l > 64 pmol/l Survivors 33 15
Fatalities 7 14
[0077] Myocardial Infarction/Prognosis
[0078] Samples were obtained from 246 patients with acute heart
attacks three days after the heart attack occurred, and NT-proET-1
was measured. The patients were monitored for a period of 60 days.
During this period, 220 patients had no adverse event, 26 passed
away or were hospitalized again due to cardiac insufficiency. Using
the receiver-operating characteristic analysis (AUC=0.70), the best
cut-off value (defined as the largest product of sensitivity and
specificity) was determined for the prognosis of the mortality rate
or rehospitalization due to cardiac insufficiency: 63.9 pmol/l. At
this cut-off value, the sensitivity of the prognosis was 65.4%, the
specificity was 73.6%. The likelihood ratio of an adverse event at
a cut-off value of 63.9 pmol/l was 2.5.
TABLE-US-00003 <63.9 pmol/l >63.9 pmol/l No adverse event 162
58 Fatalities/cardiac 9 17 insufficiency
[0079] Pneumonia/Severity and Prognosis
[0080] Samples were obtained from 142 patients with
community-associated pneumonia upon admission to the hospital, and
NT-proET-1 was measured. The patients were monitored for a period
of 70 days. During this period, 10 patients passed away. NT-proET-1
concentrations rose with the PSI (pneumonia severity index), a
score of the severity of the disease (FIG. 3), and at 53 pmol/l
they were on average higher than in the healthy control patients
(30.5 pmol/l). For the prognosis of mortality, the
receiver-operating characteristic analysis showed an AUC of 0.89.
Using the receiver-operating characteristic analysis, the best
cut-off value (defined as the largest product of sensitivity and
specificity) was determined for the prognosis of the mortality
rate: 77 pmol/l. At this cut-off value, the sensitivity of the
prognosis was 100%, the specificity was 81.2%. The likelihood ratio
of mortality at a cut-off value of 77 pmol/l was 5.5.
TABLE-US-00004 <77 pmol/l > 77 pmol/l No adverse event 108 24
Fatalities 0 10
[0081] Exacerbated COPD
[0082] NT-proET-1 was measured for 53 patients with chronic
obstructive pulmonary disease and simultaneous infection of the
lower respiratory tract. With a mean of 47 pmol/l, these patients
had higher values compared to the healthy control patients (30.5
pmol/l), however lower than the pneumonia patients (see above 53
pmol/l).
TABLE-US-00005 TABLE Classification of the New York Heart
Association (NHYA) NYHA I No limitation of physical activity.
Ordinary physical activity does not cause undue fatigue,
dysrhythmia, dyspnea or angina pectoris. NYHA II Slight limitation
of physical activity. Comfortable at rest, but ordinary physical
activity results in fatigue, dysrhythmia, dyspnea or angina
pectoris. NYHA III Marked limitation of physical activity.
Comfortable at rest, but less than ordinary physical activity
causes fatigue, dysrhythmia, dyspnea or angina pectoris. NYHA IV
Unable to carry out any physical activity without discomfort and
discomfort at rest. Bedridden.
DESCRIPTION OF THE FIGURES
[0083] FIG. 1 shows the amino acid sequence (AS) of
preproEndothelin-1 with sections 1-17 signal peptide, 18-52
NT-proET-1, 53-73 mature ET-1, 53-90 Big-ET-1 (or 74-90).
[0084] FIG. 2:
[0085] NT-proET-1 concentrations as a function of the severity of
cardiac insufficiency. Illustrated are the mean values (+SEM) of
healthy control patients (n=200) and of cardiac insufficiency
patients grouped based on the NYHA severity score (NYHA I: n=22,
NYHA II: n=126, NYHA III: n=132, NYHA 4: n=17).
[0086] FIG. 3: NT-proET-1 concentrations as a function of the
severity of pneumonia. Illustrated are the mean values (+SEM) of
healthy control patients (n=200) and of pneumonia patients grouped
based on the Pneumonia Severity Index (PSI I: n=36, PSI II: n=44,
PSI III: n=25, PSI IV: n=19, PSI V: n=18).
Sequence CWU 1
1
61212PRTHomo sapiens 1Met Asp Tyr Leu Leu Met Ile Phe Ser Leu Leu
Phe Val Ala Cys Gln 1 5 10 15 Gly Ala Pro Glu Thr Ala Val Leu Gly
Ala Glu Leu Ser Ala Val Gly 20 25 30 Glu Asn Gly Gly Glu Lys Pro
Thr Pro Ser Pro Pro Trp Arg Leu Arg 35 40 45 Arg Ser Lys Arg Cys
Ser Cys Ser Ser Leu Met Asp Lys Glu Cys Val 50 55 60 Tyr Phe Cys
His Leu Asp Ile Ile Trp Val Asn Thr Pro Glu His Val 65 70 75 80 Val
Pro Tyr Gly Leu Gly Ser Pro Arg Ser Lys Arg Ala Leu Glu Asn 85 90
95 Leu Leu Pro Thr Lys Ala Thr Asp Arg Glu Asn Arg Cys Gln Cys Ala
100 105 110 Ser Gln Lys Asp Lys Lys Cys Trp Asn Phe Cys Gln Ala Gly
Lys Glu 115 120 125 Leu Arg Ala Glu Asp Ile Met Glu Lys Asp Trp Asn
Asn His Lys Lys 130 135 140 Gly Lys Asp Cys Ser Lys Leu Gly Lys Lys
Cys Ile Tyr Gln Gln Leu 145 150 155 160 Val Arg Gly Arg Lys Ile Arg
Arg Ser Ser Glu Glu His Leu Arg Gln 165 170 175 Thr Arg Ser Glu Thr
Met Arg Asn Ser Val Lys Ser Ser Phe His Asp 180 185 190 Pro Lys Leu
Lys Gly Lys Pro Ser Arg Glu Arg Tyr Val Thr His Asn 195 200 205 Arg
Ala His Trp 210 214PRTHomo sapiens 2Ala Pro Glu Thr Ala Val Leu Gly
Ala Glu Leu Ser Ala Val 1 5 10 321PRTHomo sapiens 3Gly Glu Asn Gly
Gly Glu Lys Pro Thr Pro Ser Pro Pro Trp Arg Leu 1 5 10 15 Arg Arg
Ser Lys Arg 20 415PRTArtificialsynthetic peptide 4Cys Ala Pro Glu
Thr Ala Val Leu Gly Ala Glu Leu Ser Ala Val 1 5 10 15
522PRTartificialsynthetic peptide 5Gly Glu Asn Gly Gly Glu Lys Pro
Thr Pro Ser Pro Pro Trp Arg Leu 1 5 10 15 Arg Arg Ser Lys Arg Cys
20 635PRTHomo sapiens 6Ala Pro Glu Thr Ala Val Leu Gly Ala Glu Leu
Ser Ala Val Gly Glu 1 5 10 15 Asn Gly Gly Glu Lys Pro Thr Pro Ser
Pro Pro Trp Arg Leu Arg Arg 20 25 30 Ser Lys Arg 35
* * * * *
References