U.S. patent application number 12/391157 was filed with the patent office on 2009-11-05 for compositions and methods for treating cardiovascular disease and myocardial infarction with dipeptidyl peptidase inhibitors or b type natriuretic peptide analogues resistant to prolyl-specific dipeptidyl degradation.
This patent application is currently assigned to Biosite Incorporated. Invention is credited to MICHAEL WHITTAKER.
Application Number | 20090275512 12/391157 |
Document ID | / |
Family ID | 36037037 |
Filed Date | 2009-11-05 |
United States Patent
Application |
20090275512 |
Kind Code |
A1 |
WHITTAKER; MICHAEL |
November 5, 2009 |
COMPOSITIONS AND METHODS FOR TREATING CARDIOVASCULAR DISEASE AND
MYOCARDIAL INFARCTION WITH DIPEPTIDYL PEPTIDASE INHIBITORS OR B
TYPE NATRIURETIC PEPTIDE ANALOGUES RESISTANT TO PROLYL-SPECIFIC
DIPEPTIDYL DEGRADATION
Abstract
The present invention describes compositions and methods for
treating cardiovascular disease and myocardial infarction using
dipeptidyl peptidase inhibitors. Also provided are methods for
increasing natriuretic peptide function by administering one or
more analogues of B type natriuretic peptide that provide increased
stability in the presence of prolyl-specific dipeptidyl
peptide.
Inventors: |
WHITTAKER; MICHAEL; (SAN
DIEGO, CA) |
Correspondence
Address: |
WILSON, SONSINI, GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Assignee: |
Biosite Incorporated
San Diego
CA
|
Family ID: |
36037037 |
Appl. No.: |
12/391157 |
Filed: |
February 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11560425 |
Nov 16, 2006 |
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12391157 |
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10645874 |
Aug 20, 2003 |
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11560425 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
C07K 16/26 20130101;
C07K 2317/34 20130101; A61K 38/2242 20130101; G01N 33/6893
20130101; A61K 38/2242 20130101; G01N 33/74 20130101; G01N 33/68
20130101; G01N 33/543 20130101; A61K 45/06 20130101; G01N 2400/02
20130101; A61K 38/05 20130101; G01N 2440/38 20130101; G01N 33/6827
20130101; G01N 33/5306 20130101; A61K 38/06 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/22 20060101
A61K038/22; A61P 9/00 20060101 A61P009/00 |
Claims
1. A method for the treatment of cardiovascular disease comprising
administering to a subject in need of thereof, a therapeutically
effective amount of a dipeptidyl peptidase inhibitor.
2. A method for the treatment of heart failure comprising
administering to a subject in need thereof, a therapeutically
effective amount of a dipeptidyl peptidase inhibitor.
3. Use of a dipeptidyl peptidase inhibitor for the manufacture of a
medicament for the treatment of cardiovascular disease.
4. Use of a dipeptidyl peptidase inhibitor for the manufacture of a
medicament for the treatment of heart failure.
5. Use according to claim 3 or 4, or method according to claim 1 or
2, wherein the dipeptidyl peptidase inhibitor inhibits DPP-IV.
6. Use according to claim 3, or method according to claim 1,
wherein the cardiovascular disease is stroke.
7. Use according to claim 3, or method according to claim 1,
wherein the cardiovascular disease is acute myocardial
infarction.
8. Use according to claim 3, or method according to claim 1,
wherein the cardiovascular disease is systemic hypertension.
9. Use according to claim 3, or method according to claim 1,
wherein the cardiovascular disease is cardiac ischemia.
10. A pharmaceutical composition comprising a therapeutically
effective amount of a dipeptidyl peptidase inhibitor in combination
with one or more pharmaceutically acceptable carriers for the
treatment of cardiovascular disease.
11. A pharmaceutical composition comprising a therapeutically
effective amount of a dipeptidyl peptidase inhibitor in combination
with one or more pharmaceutically acceptable carriers for the
treatment of heart failure.
12. A pharmaceutical composition according to claim 10 or 11,
wherein the dipeptidyl peptidase inhibitor inhibits DPP-IV.
13. A method of treating a subject in need of increased natriuretic
peptide function, comprising: Administering one or more analogues
of B-type natriuretic peptide that provide increased stability in
the presence of prolyl-specific DPP, relative to B-type natriuretic
peptide.
14. A method according to claim 13, wherein said subject suffers
from one or more conditions selected from the group consisting of
stroke, congestive heart failure, cardiac ischemia, systemic
hypertension, and myocardial infarction.
15. A method according to claim 13, further comprising
administering one or more inhibitors of prolyl-specific DPP to said
subject.
16. A method according to claim 13, further comprising
administering one or more inhibitors of neutral endopeptidase to
said subject.
17. A method according to claim 15, further comprising
administering one or more inhibitors of neutral endopeptidase to
said subject.
Description
[0001] This application is a continuation of Ser. No. 10/938,760
filed Sep. 9, 2004, which is a continuation-in-part of U.S. patent
application Ser. No. 10/645,874 filed Aug. 20, 2003, which claims
priority to U.S. Provisional Patent Application No. 60/642,086
filed Feb. 4, 2004, from each of which priority is claimed, and
each of which is hereby incorporated by reference in its entirety,
including all tables, figures, and claims.
FIELD OF THE INVENTION
[0002] The present invention relates to medical diagnostics and
therapeutics.
BACKGROUND OF THE INVENTION
[0003] The following discussion of the background of the invention
is merely provided to aid the reader in understanding the invention
and is not admitted to describe or constitute prior art to the
present invention.
[0004] Natriuretic peptides are a group of naturally occurring
substances that act in the body to oppose the activity of the
renin-angiotensin system. There are three major natriuretic
peptides: atrial natriuretic peptide (ANP), which is synthesized in
the atria; brain-type natriuretic peptide (BNP), which is
synthesized in the ventricles; and C-type natriuretic peptide
(CNP), which is synthesized in the brain.
[0005] Mature human A-type natriuretic peptide (ANP) (also referred
to as atrial natriuretic peptide) is a biologically active 28 amino
acid peptide that is synthesized, stored, and released by atrial
myocytes in response to atrial distension, angiotensin II
stimulation, endothelin, and sympathetic stimulation
(beta-adrenoceptor mediated). Mature ANP is generated by
proteolytic cleavage of a 128 amino acid precursor molecule
(pro-ANP), yielding the biologically active 28 amino acid peptide
representing amino acids 99-126 of the pro-ANP molecule
(ANP.sub.99-126). Linear peptide fragments from the N-terminal
prohormone segment have also been reported to have biological
activity.
[0006] Mature human B-type natriuretic peptide (BNP) (also called
brain-type natriuretic peptide) is a 32 amino acid, 4 kDa
biologically active peptide that is involved in the natriuresis
system to regulate blood pressure and fluid balance (Bonow, R. O.,
Circulation 93:1946-1950, 1996). The mature BNP hormone is
generated by proteolytic cleavage of a 108-amino acid precursor
molecule, referred to herein as "pro-BNP." Cleavage generates t a
76-amino acid N-terminal peptide (amino acids 1-76), referred to as
"NT pro BNP," and the 32-amino acid mature hormone, referred to as
BNP or BNP.sub.32 (amino acids 77-108). It has been suggested that
each of these species--NT pro-BNP, BNP-32, and the pre-pro-BNP--can
circulate in human plasma (Tateyama et al., Biochem. Biophys. Res.
Commun. 185:760-7, 1992; Hunt et al., Biochem. Biophys. Res.
Commun. 214:1175-83, 1995).
[0007] Mature human C-type natriuretic peptide (CNP) a 22-amino
acid peptide that is the primary active natriuretic peptide in the
human brain; CNP is also considered to be an endothelium-derived
relaxant factor, which acts in the same way as nitric oxide (NO)
(Davidson et al., Circulation 93:1155-9, 1996). CNP is structurally
related to A-type natriuretic peptide (ANP) and B-type natriuretic
peptide (BNP); however, while ANP and BNP are synthesized
predominantly in the myocardium, CNP is synthesized in the vascular
endothelium as a precursor (pro-CNP) (Prickett et al., Biochem.
Biophys. Res. Commun. 286:513-7, 2001). CNP is thought to possess
vasodilator effects on both arteries and veins and has been
reported to act mainly on the vein by increasing the intracellular
cGMP concentration in vascular smooth muscle cells.
[0008] ANP and BNP are released in response to atrial and
ventricular stretch, respectively, and will cause vasorelaxation,
inhibition of aldosterone secretion in the adrenal cortex, and
inhibition of renin secretion in the kidney. Both ANP and BNP will
cause natriuresis and a reduction in intravascular volume, effects
amplified by the antagonism of antidiuretic hormone (ADH). The
physiologic effects of CNP differ from those of ANP and BNP; CNP
has a hypotensive effect, but no significant diuretic or
natriuretic actions. Increased blood levels of natriuretic peptides
have been found in certain disease states, suggesting a role in the
pathophysiology of those diseases, including stroke, congestive
heart failure (CHF), cardiac ischemia, systemic hypertension, and
acute myocardial infarction. See, e.g., WO 02/089657; WO 02/083913;
and WO 03/016910, each of which is hereby incorporated in its
entirety, including all tables, figures, and claims. Numerous
non-human homologs of the natriuretic peptides are known to those
of skill in the art.
[0009] The natriuretic peptides, alone, collectively, and/or
together with additional proteins, can serve as disease markers and
indicators of prognosis in various cardiovascular conditions. For
example, BNP, which is synthesized in the cardiac ventricles and
correlates with left ventricular pressure, amount of dyspnea, and
the state of neurohormonal modulation, makes this peptide the first
potential marker for heart failure. Measurement of plasma BNP
concentration is evolving as a very efficient and cost effective
mass screening technique for identifying patients with various
cardiac abnormalities regardless of etiology and degree of LV
systolic dysfunction that can potentially develop into obvious
heart failure and carry a high risk of a cardiovascular event.
Finding a simple blood test that would aid in the diagnosis and
management of patients with CHF clearly would have a favorable
impact on the staggering costs associated with the disease.
[0010] Removal of the natriuretic peptides from the circulation is
affected mainly by binding to clearance receptors and enzymatic
degradation in the circulation. See, e.g., Cho et al.,Heart Dis. 1:
305-28, 1999; Smith et al., J. Endocrinol. 167: 239-46, 2000.
Additionally, human pro-BNP is reported to be processed in serum
such that circulating pre-pro-BNP is unlikely to be the intact 108
amino acid form. Hunt et al., Peptides 18: 1475-81, 1997.
Degradation of the natriuretic peptides is believed mediated by
neutral endopeptidase. For example, Norman et al. (Biochem.
Biophys. Res. Commun. 28:175: 22-30, 1991) report that neutral
endopeptidase can cleave human BNP between residues 2 and 3,
between residues 4 and 5, and between residues 17 and 18.
Similarly, Lindberg and Andersson (Regul. Pept. 47: 53-63, 1993)
report that human ANP is cleaved between residues 3 and 4 and
residues 14 and 15. The biological activity of this hydrolyzed
product was about 500-fold less than intact ANP. Additionally,
Knecht et al. (Life Sci. 71: 2701-12, 2002) report that renal
neutral endopeptidase is upregulated in heart failure, a condition
where natriuretic peptide levels are increased. For this reason,
neutral endopeptidase has been targeted for inhibition in treatment
of cardiovascular disease. See, e.g., Corti et al., Circulation
104: 1856-62, 2001.
[0011] Confusion over the stability of the natriuretic peptides,
particularly in blood-derived samples (e.g., serum, plasma, whole
blood) has been reported. ANP is reported to be a better substrate
for neutral endopeptidase than is BNP. Similarly, Shimizu et al.
(Clin. Chem. Acta 305: 181-6, 2001), Gobinet-Georges et al. (Clin.
Chem. Lab. Med. 38: 519-23, 2000) and Murdoch et al. (Heart 78:
594-7, 1997) report that BNP is stable in certain blood-derived
samples or when blood is collected under certain conditions. A more
recent report by Shimizu et al. (Clin. Chem. Acta 316: 129-35,
2002) indicates that 94% of BNP in whole blood was a digested form
in which 2 amino terminal residues had been removed; and that BNP
in plasma was degraded to a number of unidentified forms.
BRIEF SUMMARY OF THE INVENTION
[0012] The present invention relates in part to compositions and
methods designed to determine the presence or amount of
biologically active natriuretic peptides, or their fragments, in a
sample. The degradation of natriuretic peptides is an ongoing
process that may be a function of, inter alia, the elapsed time
between onset of an event triggering natriuretic peptide release
into the tissues and the time the sample is obtained or analyzed;
the quantity of proteolytic enzymes present; etc. This degradation
can produce circulating amounts of natriuretic peptides having
reduced or lost biological function (referred to herein for
convenience as "inactive fragments" of a natriuretic peptide).
[0013] Failure to consider this degradation when designing an assay
for one or more natriuretic peptides may result in an assay that
detects both biologically active forms of a natriuretic peptide(s)
of interest, as well as inactive fragments of the natriuretic
peptide(s). This may lead to the conclusion that an assay shows
particularly good stability (i.e., the analyte of interest is not
lost to the assay during sample storage), when in fact the
natriuretic peptide of interest is actually being degraded to an
inactive fragment and the assay result is confounded by the
inability to distinguish the intended analyte from the pool of
inactive fragments originally present in the sample. Because the
biologically active forms may be more relevant to the physiologic
state of the subject, and because upregulated proteolytic enzymes
in diseased subjects may lead to particularly large pools of
inactive fragments in the subjects of potentially the greatest
interest, the compositions and methods described herein may provide
improved diagnostic and prognostic information to the artisan in
comparison to assays that are not specific for the biologically
active forms.
[0014] The methods and compositions described herein can meet the
need in the art for rapid, sensitive and specific diagnostic assay
to be used in the diagnosis and differentiation of various
cardiovascular diseases, including stroke, congestive heart failure
(CHF), cardiac ischemia, systemic hypertension, and/or acute
myocardial infarction. Moreover, the methods and compositions of
the present invention can also be used to facilitate the treatment
of patients and the development of additional diagnostic and/or
prognostic indicators and indicator panels.
[0015] In a first aspect then, the present invention relates to
methods for detecting the presence or amount of a natriuretic
peptide in a sample, comprising performing an assay that detects a
biologically active natriuretic peptide, but that exhibits at least
a 5-fold reduction in signal from, and preferably does not
appreciably detect, one or more biologically inactive fragments of
the natriuretic peptide. Biologically inactive fragments may
include those in which residues from either or both of the
N-terminus or C-terminus of the biologically active natriuretic
peptide have been removed, and/or in which the loop formed by
intramolecular disulfide bonding of the natriuretic peptide has
been cleaved. Such biologically inactive fragments may be formed,
for example, by cleaving one or more peptide bonds in the
biologically active natriuretic peptide.
[0016] In related aspects, the present invention relates to methods
for detecting the presence or amount of a natriuretic peptide in a
sample, comprising performing an assay that detects an intact
natriuretic peptide, but that exhibits at least a 5-fold reduction
in signal from, and preferably does not appreciably detect, an
equimolar amount of a peptide that is generated when a portion, and
preferably at least an N-terminal portion, of the intact
natriuretic peptide is removed.
[0017] In various embodiments, the present invention relates to
methods for detecting the presence or amount of BNP in a sample,
comprising performing an assay that detects BNP.sub.77-108, but
that exhibits at least a 5-fold reduction in signal from, and
preferably does not appreciably detect, an equimolar amount of
BNP.sub.94-108; the assay detects BNP.sub.77-108, but exhibits at
least a 5-fold reduction in signal from, and preferably does not
appreciably detect, BNP.sub.90-108; the assay detects
BNP.sub.77-108, but exhibits at least a 5-fold reduction in signal
from, and preferably does not appreciably detect, BNP.sub.81-108;
the assay detects BNP.sub.77-108, but exhibits at least a 5-fold
reduction in signal from, and preferably does not appreciably
detect, BNP.sub.79-108; the assay detects BNP.sub.77-108, but
exhibits at least a 5-fold reduction in signal from, and preferably
does not appreciably detect, BNP.sub.77-106; and/or the assay
detects BNP.sub.77-108, but exhibits at least a 5-fold reduction in
signal from, and preferably does not appreciably detect,
BNP.sub.79-106.
[0018] In various additional embodiments, the present invention
relates to methods for detecting the presence or amount of BNP in a
sample, comprising performing an assay that detects BNP.sub.1-76,
but that exhibits at least a 5-fold reduction in signal from, and
preferably does not appreciably detect, an equimolar amount of
BNP.sub.38-76; the assay detects BNP.sub.1-76, but exhibits at
least a 5-fold reduction in signal from, and preferably does not
appreciably detect, BNP.sub.24-76; the assay detects BNP.sub.1-76,
but exhibits at least a 5-fold reduction in signal from, and
preferably does not appreciably detect, BNP.sub.12-76; the assay
detects BNP.sub.1-76, but exhibits at least a 5-fold reduction in
signal from, and preferably does not appreciably detect,
BNP.sub.3-76; the assay detects BNP.sub.1-76, but exhibits at least
a 5-fold reduction in signal from, and preferably does not
appreciably detect, BNP.sub.1-73; and/or the assay detects
BNP.sub.1-76, but exhibits at least a 5-fold reduction in signal
from, and preferably does not appreciably detect, BNP.sub.3-73.
[0019] In still other additional embodiments, the present invention
relates to methods for detecting the presence or amount of ANP in a
sample, comprising performing an assay that detects ANP.sub.99-126,
but that exhibits at least a 5-fold reduction in signal from, and
preferably does not appreciably detect, an equimolar amount of
ANP.sub.113-126; the assay detects ANP.sub.99-126, but exhibits at
least a 5-fold reduction in signal from, and preferably does not
appreciably detect, ANP.sub.105-126; the assay detects
ANP.sub.99-126, but exhibits at least a 5-fold reduction in signal
from, and preferably does not appreciably detect, ANP.sub.102-126;
the assay detects ANP.sub.99-126, but exhibits at least a 5-fold
reduction in signal from, and preferably does not appreciably
detect, ANP.sub.99-124; and/or the assay detects ANP.sub.99-126,
but exhibits at least a 5-fold reduction in signal from, and
preferably does not appreciably detect, ANP.sub.102-124.
[0020] As described hereinafter, such assays may be designed in a
variety of ways known to those of skill in the art. Preferred
assays are immunoassays, although other methods are well known to
those skilled in the art (for example, the use of biosensors, or
the use of natural receptors for natriuretic peptides that are
known in the art). Any suitable immunoassay may be utilized, for
example, assays which directly detect analyte binding (e.g., by
ellipsometric detection), enzyme-linked immunoassays (ELISA),
radioimmunoassays (RIAs), competitive binding assays, sandwich
immunoassays, and the like. Specific immunological binding of the
antibody to the one or more natriuretic peptide fragments can be
detected directly or indirectly. Direct labels include fluorescent
or luminescent tags, metals, dyes, radionuclides, and the like,
attached to the antibody. Indirect labels include various enzymes
well known in the art, such as alkaline phosphatase, horseradish
peroxidase and the like. Antibodies attached to a second molecule,
such as a detectable label, are referred to herein as "antibody
conjugates." The skilled artisan will also understand that natural
receptors for the natriuretic peptides exist, and that these
receptors may also be used in a manner akin to antibodies in
providing binding assays.
[0021] Immunoassays may be formulated using one or more antibodies
selected to bind to an epitope that is partially or completely lost
from biologically inactive fragments of the natriuretic peptide as
compared to the intact natriuretic peptide. For example, in a
sandwich assay, if an antibody bound to a solid phase is selected
to bind preferentially to the N-terminal portion of the molecule,
and a labeled antibody is selected to bind to the C-terminal
portion of the molecule, only those molecules that contain both the
N- and C-terminal portions of the molecule will be detected in the
assay. Alternatively, both the solid phase and labeled antibodies
may be selected to bind to the N-terminal portion of the
molecule.
[0022] The skilled artisan will understand that cleavage of the
natriuretic peptide may remove all of the epitope to which an
antibody binds (e.g., the antibody binds to the N-terminal region
alone). Alternatively, an epitope may be formed from portions of
the natriuretic peptide that are not contiguous in the linear
sequence of the molecule, but that are associated in 3-dimensional
space in solution, so that epitope comprises more than the
described amino acid residues, but removal of the region described
amino acid residues results in reduced binding of the antibody, and
hence a loss of signal in the assay.
[0023] In certain embodiments, antibodies are selected, based not
upon a particular affinity for one or more natriuretic peptide(s),
but instead based upon a signal that is obtainable in a binding
assay such as an immunoassay. The skilled artisan will recognize
that various binding assay formats are known in the art, and that
it is often the use of antibodies to formulate an appropriate assay
that is more important than a particular affinity of an antibody
for one or more target molecules. For example, competitive binding
assays may comprise a receptor (e.g., an antibody) bound to a solid
surface. An analyte of interest in a test sample competes for
binding with a labeled molecule that also binds to the receptor.
The amount of labeled molecule bound to the receptor (and hence
assay signal) is inversely proportional to the amount of analyte of
interest in the test sample. In this case, a single antibody
attached to the solid phase is used. Alternatively, in a sandwich
immunoassay, a first antibody, typically bound to a solid surface,
and a second antibody, typically conjugated to a detectable label,
each bind to an analyte of interest in a test sample. The amount of
labeled molecule bound to the receptor (and hence assay signal) is
directly proportional to the amount of analyte of interest in the
test sample.
[0024] The immunoassays of the present invention are preferably
designed to distinguish a biologically active natriuretic peptide
from a biologically inactive natriuretic peptide and/or an intact
natriuretic peptide from a natriuretic peptide fragment. For
example, a preferred immunoassay would distinguish a natriuretic
peptide comprising an intact N-terminal region from a fragment of
the natriuretic peptide from which the N-terminal region has been
lost. An immunoassay is said to "distinguish" between a first group
of polypeptides and a second group of polypeptides if the
immunoassay provides a signal related to binding of the first group
of polypeptides that is at least a factor of 5 greater than a
signal obtained from an equal number of molecules of the second
group of polypeptides under the same assay conditions, when the
assay is performed at no more than twice the amount of the first
group of polypeptides necessary to obtain a maximum signal. More
preferably, the signal is at least a factor of 10 greater, even
more preferably at least a factor of 20 greater, and most
preferably at least a factor of 50 greater, at least a factor of
100 greater, or more under such assay conditions. An assay does not
"appreciably detect" the second group of polypeptides if a signal
related to binding of the first group of polypeptides may be
obtained, but no signal above background is obtained from an equal
number of molecules of the second group of polypeptides under such
assay conditions.
[0025] In another aspect, the present invention relates to methods
for detecting the presence or amount of a natriuretic peptide in a
sample, comprising performing an assay in which the signal depends
upon an antibody that specifically binds to a biologically active
natriuretic peptide, but that does not specifically bind to
biologically inactive fragments of the natriuretic peptide. As
discussed above, biologically inactive fragments may include those
in which residues from either or both of the N-terminus or
C-terminus of the intact natriuretic peptide have been removed,
and/or in which the loop formed by intramolecular disulfide bonding
of the natriuretic peptide has been cleaved. In preferred
embodiments, the assay is performed under conditions in which the
signal depends upon an antibody that specifically binds to the
intact natriuretic peptide, but that does not specifically bind to
a peptide that is generated from the natriuretic peptide when an
N-terminal portion of the natriuretic peptide is removed.
[0026] In related aspects, the present invention relates to methods
for detecting the presence or amount of a natriuretic peptide in a
sample, comprising performing an assay in which the signal depends
upon an antibody that specifically binds to the intact natriuretic
peptide, but that does not specifically bind to fragments of the
natriuretic peptide generated when a portion, and preferably at
least an N-terminal portion, of the natriuretic peptide is
removed.
[0027] In various embodiments, the present invention relates to
methods for detecting the presence or amount of BNP in a sample,
comprising performing an assay in which the signal depends upon an
antibody that specifically binds to BNP.sub.77-108, but that does
not specifically bind to BNP.sub.94-108; the assay depends upon an
antibody that specifically binds to BNP.sub.77-108, but does not
specifically bind to BNP.sub.90-108; the assay depends upon an
antibody that specifically binds to BNP.sub.77-108, but does not
specifically bind to BNP.sub.81-108; the assay depends upon an
antibody that specifically binds to BNP.sub.77-108, but does not
specifically bind to BNP.sub.79-108; the assay depends upon an
antibody that specifically binds to BNP.sub.77-108, but does not
specifically bind to BNP.sub.77-106; and/or the assay depends upon
an antibody that specifically binds to BNP.sub.77-108, but does not
specifically bind to BNP.sub.79-106.
[0028] In various additional embodiments, the present invention
relates to methods for detecting the presence or amount of BNP in a
sample, comprising performing an assay in which the signal depends
upon an antibody that specifically binds to BNP.sub.1-76, but that
does not specifically bind to BNP.sub.38-76; the assay depends upon
an antibody that specifically binds to BNP.sub.1-76, but does not
specifically bind to BNP.sub.24-76; the assay depends upon an
antibody that specifically binds to BNP.sub.1-76, but does not
specifically bind to BNP.sub.12-76; the assay depends upon an
antibody that specifically binds to BNP.sub.1-76, but does not
specifically bind to BNP.sub.3-76; the assay depends upon an
antibody that specifically binds to BNP.sub.1-76, but does not
specifically bind to BNP.sub.1-73; and/or the assay depends upon an
antibody that specifically binds to BNP.sub.1-76, but does not
specifically bind to BNP.sub.3-73.
[0029] In other additional embodiments, the present invention
relates to methods for detecting the presence or amount of ANP in a
sample, comprising performing an assay in which the signal depends
upon an antibody that specifically binds to ANP.sub.99-126, but
that does not specifically bind to ANP.sub.113-126; the assay
depends upon an antibody that specifically binds to ANP.sub.99-126,
but that does not specifically bind to ANP.sub.105-126; the assay
depends upon an antibody that specifically binds to ANP.sub.99-126,
but that does not specifically bind to ANP.sub.102-126; the assay
depends upon an antibody that specifically binds to ANP.sub.99-126,
but that does not specifically bind to ANP.sub.99-124; and/or the
assay depends upon an antibody that specifically binds to
ANP.sub.99-126, but that does not specifically bind to
ANP.sub.102-124.
[0030] A signal from an immunoassay is said to "depend upon binding
to an antibody" if the antibody participates in formation of a
complex necessary to generate the signal. For example, in a
sandwich immunoassay formulated using a solid phase antibody and a
second antibody conjugate, each of which must bind to an analyte to
form the sandwich, each of the solid phase antibody and second
antibody participate in formation of the complex necessary to
generate the signal. In a competitive immunoassay where a single
antibody is used, and an analyte competes with an analyte conjugate
for binding, the single antibody participates in formation of the
complex necessary to generate the signal. The skilled artisan will
understand that numerous additional immunoassay formulations may be
provided.
[0031] The assay methods described herein may also comprise a step
of storing a sample for a period of time prior to assay for one or
more natriuretic peptides. Because degradation of natriuretic
peptides may be an ongoing process during storage, the storage
considerations should be selected to reduce loss of the N-terminal
portion of the molecule. Thus, the storage conditions may comprise
addition of one or more inhibitors of natriuretic peptide
degradation. As discussed hereinafter, the storage conditions may
comprise one or more inhibitors of neutral endopeptidase and/or one
or more inhibitors of prolyl-specific dipeptidyl peptidase. Such
inhibitors are well known in the art. See, e.g., Corti et
al.,Circulation 104: 1856-62, 2001; Senten et al., J. Comb. Chem.
5: 336-44, 2003; Senten et al., Bioorg. Med. Chem. Lett. 12:
2825-8, 2002. In an alternative or in conjunction with such
inhibitors, storage conditions may comprise storage at a reduced
temperature, preferably below the freezing point of the sample.
[0032] In another aspect, the present invention relates to an assay
device configured and arranged to perform the described assays.
Devices for performing the assays described herein preferably
contain a plurality of discrete, independently addressable
locations, or "diagnostic zones," each of which is related to a
particular analyte or set of analytes of interest, one or more of
which is a natriuretic peptide. For example, each of a plurality of
discrete zones may comprise a receptor (e.g., an antibody) for
binding a different analyte. Following reaction of a sample with
the devices, a signal is generated from the diagnostic zone(s),
which may then be correlated to the presence or amount of the
peptide of interest.
[0033] In yet another aspect, the presence or amount of one or more
natriuretic peptide(s) of interest measured by the methods
described herein may be related to the presence or absence of a
disease, or a disease prognosis (e.g., the likelihood of a future
adverse outcome related to a disease). Preferred diseases include
various cardiovascular and cerebrovascular diseases, including
stroke, congestive heart failure (CHF), cardiac ischemia, systemic
hypertension, and/or acute myocardial infarction. These methods
preferably comprise determining the presence or amount of one or
more natriuretic peptide(s) by the methods described herein, and
relating that presence or amount to the disease or prognosis of
interest.
[0034] In certain embodiments, the signal obtained from an assay
need not be related to the presence or amount of one or more
natriuretic peptide(s); rather, the signal may be directly related
to the presence or absence of a disease, or the likelihood of a
future adverse outcome related to a disease. For example, a level
of signal x may indicate that y pg/mL of a natriuretic peptide is
present in the sample. A table may then indicate that y pg/mL of
that natriuretic peptide indicates congestive heart failure. It may
be equally valid to simply relate a level of signal x directly to
congestive heart failure, without determining how much of the
natriuretic peptide is present. Such a signal is preferably
obtained from an immunoassay using the antibodies of the present
invention, although other methods are well known to those skilled
in the art.
[0035] In still another aspect, the present invention relates to
methods for selecting one or more antibodies for use in an assay
for natriuretic peptide(s). These methods comprise selecting
antibodies that, when used in an assay, detect a biologically
active natriuretic peptide of interest, but that exhibit at least a
5-fold reduction in signal from, and preferably do not appreciably
detect, biologically inactive fragments of the natriuretic peptide.
As above, biologically inactive fragments may include those in
which residues from either or both of the N-terminus or C-terminus
of the intact natriuretic peptide have been removed, and/or in
which the loop formed by intramolecular disulfide bonding of the
natriuretic peptide has been cleaved.
[0036] In related aspects, the present invention relates to methods
for selecting one or more antibodies for use in an assay,
comprising selecting antibodies that, when used in an assay, detect
an intact natriuretic peptide of interest, but that exhibit at
least a 5-fold reduction in signal from, and preferably do not
appreciably detect, an equimolar amount of a peptide that is
generated from the natriuretic peptide when a portion, and
preferably an N-terminal portion, of the natriuretic peptide is
removed.
[0037] In various embodiments, the methods comprise selecting one
or more antibodies that detect BNP.sub.77-108 when used in an
assay, but that exhibit at least a 5-fold reduction in signal from,
and preferably do not appreciably detect, an equimolar amount of
BNP.sub.94-108; selecting one or more antibodies that detect
BNP.sub.77-108, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect,
BNP.sub.90-108; selecting one or more antibodies that detect
BNP.sub.77-108, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect,
BNP.sub.81-108; selecting one or more antibodies that detect
BNP.sub.77-108, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect,
BNP.sub.79-108; selecting one or more antibodies that detect
BNP.sub.77-108, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect,
BNP.sub.77-106; and/or selecting one or more antibodies that detect
BNP.sub.77-108, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect,
BNP.sub.79-106.
[0038] In various additional embodiments, the methods comprise
selecting one or more antibodies that detect BNP.sub.1-76, when
used in an assay, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect, an equimolar
amount of BNP.sub.38-76; selecting one or more antibodies that
detect BNP.sub.1-76, but that exhibit at least a 5-fold reduction
in signal from, and preferably do not appreciably detect,
BNP.sub.24-76; selecting one or more antibodies that detect
BNP.sub.1-76, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect,
BNP.sub.12-76; selecting one or more antibodies that detect
BNP.sub.1-76, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect,
BNP.sub.3-76; selecting one or more antibodies that detect
BNP.sub.1-76, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect,
BNP.sub.1-73; and/or selecting one or more antibodies that detect
BNP.sub.1-76, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect,
BNP.sub.3-73.
[0039] In other additional embodiments, the methods comprise
selecting one or more antibodies that detect ANP.sub.99-126 when
used in an assay, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect, an equimolar
amount of ANP.sub.113-126; selecting one or more antibodies that
detect ANP.sub.99-126 when used in an assay, but that exhibit at
least a 5-fold reduction in signal from, and preferably do not
appreciably detect, an equimolar amount of ANP.sub.105-126;
selecting one or more antibodies that detect ANP.sub.99-126 when
used in an assay, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect, an equimolar
amount of ANP.sub.101-126; selecting one or more antibodies that
detect ANP.sub.99-126 when used in an assay, but that exhibit at
least a 5-fold reduction in signal from, and preferably do not
appreciably detect, an equimolar amount of ANP.sub.99-124; and/or
selecting one or more antibodies that detect ANP.sub.99-126 when
used in an assay, but that exhibit at least a 5-fold reduction in
signal from, and preferably do not appreciably detect, an equimolar
amount of ANP.sub.101-124.
[0040] In other related aspects, the present invention relates to a
method of selecting one or more antibodies for use in an assay for
natriuretic peptide(s). The methods comprise selecting one or more
antibodies that specifically bind to a biologically active
natriuretic peptide, but that do not specifically bind to
biologically inactive fragments of the natriuretic peptide. As
discussed above, biologically inactive fragments may include those
in which residues from either or both of the N-terminus or
C-terminus of the intact natriuretic peptide have been removed,
and/or in which the loop formed by intramolecular disulfide bonding
of the natriuretic peptide has been cleaved. In preferred
embodiments, the assay is performed under conditions in which the
signal depends upon an antibody that specifically binds to the
intact natriuretic peptide, but that does not specifically bind to
a peptide that is generated from the natriuretic peptide when an
N-terminal portion of the natriuretic peptide is removed
[0041] In still other related aspects, the present invention
relates to methods for selecting one or more antibodies for use in
an assay, comprising selecting antibodies that specifically bind to
the intact natriuretic peptide, but that do not specifically bind
to biologically inactive fragments of the natriuretic peptide
generated when an N-terminal portion of the natriuretic peptide is
removed.
[0042] In various embodiments, the methods comprise selecting one
or more antibodies that specifically bind to BNP.sub.77-108, but
that do not specifically bind to BNP.sub.94-108; selecting one or
more antibodies that specifically bind to BNP.sub.77-108, but that
do not specifically bind to BNP.sub.90-108; selecting one or more
antibodies that specifically bind to BNP.sub.77-108, but that do
not specifically bind to BNP.sub.81-108; selecting one or more
antibodies that specifically bind to BNP.sub.77-108, but that do
not specifically bind to BNP.sub.79-108; selecting one or more
antibodies that specifically bind to BNP.sub.77-108, but that do
not specifically bind to BNP.sub.77-106; and/or selecting one or
more antibodies that specifically bind to BNP.sub.77-108, but that
do not specifically bind to BNP.sub.79-106.
[0043] In various additional embodiments, the methods comprise
selecting one or more antibodies that specifically bind to
BNP.sub.1-76, but that do not specifically bind to BNP.sub.38-76;
selecting one or more antibodies that specifically bind to
BNP.sub.1-76, but that do not specifically bind to BNP.sub.24-76;
selecting one or more antibodies that specifically bind to
BNP.sub.1-76, but that do not specifically bind to BNP.sub.12-76;
selecting one or more antibodies that specifically bind to
BNP.sub.1-76, but that do not specifically bind to BNP.sub.3-76;
selecting one or more antibodies that specifically bind to
BNP.sub.1-76, but that do not specifically bind to BNP.sub.1-73;
and/or selecting one or more antibodies that specifically bind to
BNP.sub.1-76, but that do not specifically bind to
BNP.sub.3-73.
[0044] In other additional embodiments, the methods comprise
selecting one or more antibodies that specifically bind to
ANP.sub.99-126, but that do not specifically bind to
ANP.sub.113-126; selecting one or more antibodies that specifically
bind to ANP.sub.99-126, but that do not specifically bind to
ANP.sub.105-126; selecting one or more antibodies that specifically
bind to ANP.sub.99-126, but that do not specifically bind to
ANP.sub.101-26; selecting one or more antibodies that specifically
bind to ANP.sub.99-126, but that do not specifically bind to
ANP.sub.99-124; and/or selecting one or more antibodies that
specifically bind to ANP.sub.99-126, but that do not specifically
bind to ANP.sub.101-124.
[0045] In another aspect, one or more antibodies and/or antibody
conjugates of the present invention may be provided as kits for
determining the presence or amount of natriuretic peptide(s). These
kits preferably comprise devices and reagents for performing at
least one assay as described herein on a test sample. Such kits
preferably contain sufficient reagents to perform one or more such
determinations, and/or Food and Drug Administration (FDA)-approved
labeling.
[0046] In still another aspect, the invention relates to methods
for determining a treatment regimen for use in a patient. The
methods preferably comprise determining the presence or amount of
one or more natriuretic peptide(s) by the methods described herein,
and relating this presence or amount to a disease or prognostic
state. As discussed herein, diagnosis and differentiation of
various cardiovascular and cerebrovascular diseases, including
stroke, congestive heart failure (CHF), cardiac ischemia, systemic
hypertension, acute coronary syndrome, and/or acute myocardial
infarction may be related to ANP, BNP, and/or CNP levels. Once a
diagnosis or prognosis is obtained, a treatment regimen is selected
to be consistent with that diagnosis.
[0047] It is another object of the invention to provide
compositions and methods for stabilizing natriuretic peptides. Such
methods may improve the therapeutic potential of natriuretic
peptides, particularly for the treatment of cardiovascular
diseases. Several natriuretic peptides, including pro-BNP, mature
BNP, and pro-ANP comprise a penultimate proline residue, and are
suitable substrates for prolyl-specific dipeptidyl dipeptidases
("DPPs"). Thus, while mature BNP has been reported to exhibit
resistance to degradation by neutral endopeptidase relative to ANP,
DPPs may represent a previously unrecognized degradation pathway
for the mature BNP molecule as well as for pro-BNP and pro-ANP.
Furthermore, the removal of the proline-containing dipeptide may
open the various natriuretic peptides to further degradation by
other peptidases. Subjects that may benefit from increased
natriuretic peptide concentrations may be treated with inhibitors
of one or more DPPs, either alone or in combination with neutral
endopeptidase inhibitors, and/or treated with natriuretic peptides
and/or natriuretic peptide analogs exhibiting increased DPP
stability. In addition, BNP in samples removed from a subject may
be stabilized during storage using these same inhibitors.
[0048] Thus, in one aspect, the present invention relates to
methods of inhibiting degradation of one or more natriuretic
peptides. The method comprises administering one or more inhibitors
of prolyl-specific DPP in an amount sufficient to inhibit
degradation of the natriuretic peptide.
[0049] In another aspect, the present invention relates to methods
for treating a subject in need of increased natriuretic peptide
function, preferably subjects suffering from heart failure. The
methods comprise administering one or more inhibitors of
prolyl-specific DPP to the subject, preferably in an amount
sufficient to inhibit degradation of the natriuretic peptide.
[0050] In certain embodiments, the inhibitor(s) of prolyl-specific
DPP are selective for one or more DPP(s) for which pro-BNP, mature
BNP, and/or pro-ANP are a substrate. Methods for designing and
selecting specific DPP inhibitors are well known in the art. See,
e.g., Leiting et al., Biochem. J. 371: 525-32, 2003; Sedo et al.,
Physiol. Res. 52: 367-72, 2003; Villhauer et al., J. Med. Chem. 46:
2774-89, 2003; Senten et al., J. Comb. Chem. 5: 336-44, 2003;
Senten et al., Bioorg. Med. Chem. Lett. 12: 2825-8, 2002; Borloo
and Meester, Verh. K. Acad. Geneeskd. Belg. 56: 57-88, 1994. In
addition, DPP may be inhibited at the level of expression by
methods known to those of skill in the art, such as by antisense or
RNAi constructs. DPPs may also be inhibited through the use of
binding proteins, e.g., antibodies or fragments thereof that
specifically bind to one or more DPPs and prevent their activity on
a natriuretic peptide substrate.
[0051] The methods described herein may comprise the use of one or
more inhibitors of prolyl-specific DPP alone, or such inhibitors
may be combined with one or more inhibitors of neutral
endopeptidase and/or other protease inhibitors, and/or with one or
more exogenously added natriuretic peptides to provide a
potentiated increase in natriuretic peptide function to the subject
in comparison to the use of inhibitors of neutral endopeptidase
and/or exogenously added natriuretic peptides in the absence of
prolyl-specific DPP inhibitor(s). These compounds may be
conveniently provided as part of a pharmaceutical composition.
[0052] In preferred embodiments, subjects receiving the treatment
methods described herein suffer from diseases selected from the
group consisting of stroke, congestive heart failure (CHF), cardiac
ischemia, systemic hypertension, and/or acute myocardial
infarction. In particularly preferred embodiments, subjects
receiving the treatment methods described herein are selected on
the basis of a BNP level. For example, subjects may be selected on
the basis of a plasma BNP level prior to receiving treatment of at
least about 80 pg/mL, preferably at least about 100 pg/mL, still
more preferably at least about 200 pg/mL, yet more preferably at
least about 500 pg/mL, and most preferably at least about 1000
pg/mL.
[0053] In yet another aspect, the present invention relates to
methods for treating a subject in need of increased natriuretic
peptide function comprising administering one or more analogues of
a natriuretic peptide that provide increased stability in the
presence of prolyl-specific DPP (e.g. as measured by an increase in
the t1/2 of the natriuretic peptide of interest in the blood of the
subject).
[0054] It is yet another object of the invention to provide methods
and compositions for determining the presence or amount of one or
more natriuretic peptides of interest, where one or more of those
natriuretic peptides of interest are glycosylated. Covalently bound
carbohydrate residues in glycosylated natriuretic peptides can have
substantial effects on the ability of various assay methods to
detect such peptides. By careful selection of assay conditions,
such effects can be mitigated, resulting in an assay result that is
representative of the presence or amount of the natriuretic
peptides of interest in a sample.
[0055] Thus, in another aspect, the present invention relates to
methods for detecting the presence or amount of one or more
natriuretic peptides of interest in a sample, where one or more of
those natriuretic peptides comprise covalently bound carbohydrate
residues. These methods comprise removing one or more covalently
bound carbohydrate residues from one or more of said natriuretic
peptides of interest, and assaying the sample for the natriuretic
peptides of interest. The assay result is thus related to the
presence or amount of said natriuretic peptides of interest in said
sample. In various embodiments, the covalently bound carbohydrate
residues may be removed from one or more naturietic peptides by
enzymatic treatment of the peptides, by non-enzymatic chemical
treatment of the peptides, or by a combination of these
methods.
[0056] Effective enzymatic methods for removing N- and O-linked
carbohydrate residues are well known in the art, using enzymes such
as N-glycanase (also known as N-glycosidase), endoglycosidase H,
endoglycosidase A, O-glycanase (also known as
endo-.alpha.-N-acetylgalactosaminidase),
.alpha.2-(3,6,8,9)-neuriminidase, .beta.(1,4)-galactosidase,
N-acetylglucosaminidase, endoglycosidase F.sub.1, endoglycosidase
F.sub.2, and/or endoglycosidase F.sub.3. This list is not meant to
be limiting.
[0057] In the case of non-enzymatic chemical treatments for removal
of covalently bound carbohydrate residues from peptides, hydrazine
hydrolysis has been found to be effective in the release of
unreduced O- and N-linked oligosaccharides. Selective and
sequential release of oligosaccharides can be accomplished by
initial mild hydrazinolysis of the O-linked oligosaccharides at
about 60.degree. C. followed by N-linked oligosaccharides at about
95.degree. C. See, e.g., Patel and Rarekh, Meth. Enzymol. 230,
58-66, 1994. Such treatment may result in destruction of the
polypeptide however. Alkaline-.beta.-elimination of O-linked
oligosaccharides, which utilizes alkaline sodium borohydride in a
mild base environment, may be preferred. See, e.g., Glycobiology. A
Practical Approach, Fukuda, M. and Kobata, A. (Eds), pp. 291-328,
IRL/Oxford Univ. Press, Oxford, 1993. In addition,
Trifluoromethanesulfonic acid hydrolysis may be employed. This
method typically leaves an intact polypeptide, but results in
destruction of the glycan. See, e.g., Edge, Biochem. J. 376:
339-50, 2003.
[0058] The foregoing methods of sugar removal from peptides may be
used on native (non-denatured) polypeptides and/or following
denaturation of the polypeptides. Whether enzymatic, non-enzymatic,
or both treatments are employed to remove covalently bound
carbohydrate residues from natriuretic peptides, it is preferred
that at least about 50%, more preferably, at least about 60%, still
more preferably at least about 70%, yet more preferably at least
about 80%, and most preferably at least about 90% to about 100% of
the carbohydrate residues are removed from one or more, and
preferably all, of the glycosylated natriuretic peptides of
interest by this treatment. The extent of glycosylation of a
polypeptide can be determined by comparing the apparent mass of the
polypeptide to the mass of the amino acid constituents of the
polypeptide, and assuming that the balance of the apparent mass is
contributed by glycosylation. In the event that other modifications
(e.g., oxidation, nitration, phosphorylation) are known to have
occurred, the mass contributed by these other modifications may
also be subtracted from the apparent mass. The extent of
carbohydrate residue removal can then be monitored by determining
the apparent mass of the polypeptide following deglycosylation
treatment. Methods for determining the apparent mass of a
polypeptide (e.g., SDS gel electrophoresis, analytical
centrifugation, gel permeation chromatography, mass spectrometry,
etc.) are well known to those of skill in the art.
[0059] The sample containing such glycosylated natriuretic peptides
may be a test sample as that term is defined herein. The
glycosylated natriuretic peptides present in such a sample may be
naturally present, such as in a sample obtained from a patient, or
may be a standard sample. Natriuretic peptides used in formulating
such standards are often expressed recombinantly in mammalian
tissue culture systems, which contain active glycosylation
functions.
[0060] Following the deglycosylation step, the methods described
herein may employ any assay methods known in the art. Such assay
methods may employ separation methods such as affinity separation,
gel electrophoresis, capillary electrophoresis, liquid
chromatography, and/or HPLC to separate analytes of interest for
detection. In preferred embodiments, immunoassay devices and
methods are often used for affinity separation, in various
sandwich, competitive, or non-competitive assay formats, to
generate a signal that is related to the presence or amount of one
or more natriuretic peptides of interest. Additionally, certain
methods and devices, such as biosensors and optical immunoassays,
may be employed to determine the presence or amount of analytes
without the need for a labeled molecule.
[0061] In addition, mass spectrometry methods may advantageously be
employed as part of the assay method. The terms "mass spectrometry"
or "MS" as used herein refer to methods of filtering, detecting,
and measuring ions based on their mass-to-charge ratio, or "m/z."
In general, one or more molecules of interest are ionized, and the
ions are subsequently introduced into a mass spectrographic
instrument where, due to a combination of magnetic and electric
fields, the ions follow a path in space that is dependent upon mass
("m") and charge ("z"). See, e.g., U.S. Pat. No. 6,204,500,
entitled "Mass Spectrometry From Surfaces;" U.S. Pat. No.
6,107,623, entitled "Methods and Apparatus for Tandem Mass
Spectrometry;" U.S. Pat. No. 6,268,144, entitled "DNA Diagnostics
Based On Mass Spectrometry;" U.S. Pat. No. 6,124,137, entitled
"Surface-Enhanced Photolabile Attachment And Release For Desorption
And Detection Of Analytes;" Wright et al., "Proteinchip surface
enhanced laser desorption/ionization (SELDI) mass spectrometry: a
novel protein biochip technology for detection of prostate cancer
biomarkers in complex protein mixtures," Prostate Cancer and
Prostatic Diseases 2: 264-76 (1999); and Merchant and Weinberger,
"Recent advancements in surface-enhanced laser
desorption/ionization-time of flight-mass spectrometry,"
Electrophoresis 21: 1164-67 (2000), each of which is hereby
incorporated by reference in its entirety, including all tables,
figures, and claims. Molecules (e.g., peptides) in a test sample
can be ionized by any method known to the skilled artisan. These
methods include, but are not limited to, electron ionization,
chemical ionization, fast atom bombardment, field desorption, and
matrix-assisted laser desorption ionization ("MALDI"), surface
enhanced laser desorption ionization ("SELDI"), photon ionization,
electrospray, and inductively coupled plasma.
[0062] In certain embodiments, the MS methods discussed above are
preferably combined with an affinity purification step such as
binding to an antibody that specifically binds one or more
polypeptides of interest. See, e.g., Nelson et al., Anal. Chem.,
67: 1153, 1995; Tubbs et al., Anal. Biochem. 289: 26, 2001.
Niederkofler et al., Anal. Chem. 73: 3294, 2001.
[0063] One feature of glycoproteins is the typical heterogeneity of
the glycans. It is very common for individual molecules of a given
glycoprotein to carry different carbohydrates at the same
attachment site in the polypeptide chain. Any structural changes in
the carbohydrate residues will result in the formation of discrete
molecular subsets referred to as glycoforms. In the case of various
separation methods, such heterogeneity can substantially complicate
the analysis due to differences in charge and mass of the various
polypeptides of interest and/or differences in the binding of the
various polypeptides of interest to a binding matrix (e.g., an
antibody). In addition, carbohydrates are not ionized as
efficiently as compounds such as proteins that can be easily
protonated; neither do they appear to be transferred to the vapor
phase as effectively.
[0064] Thus, in preferred embodiments, the methods described herein
provide an increased detection of one or more naturietic peptides
of interest, as compared to performing the same assaying step in
the absence of removing one or more covalently bound carbohydrate
residues from one or more of the natriuretic peptides of interest.
The term "increased detection" as used herein refers to an
increased signal obtained from the assay method for one or more
particular naturietic peptides of interest. Such an increased
signal may be representative of an increased ability to detect all
of the naturietic peptides of interest. For example, an antibody
that could not bind certain glycosylated forms of one or more
naturietic peptides of interest would result in an assay signal
that underestimates the concentration of those naturietic peptides;
or less efficient ionization of certain glycosylated forms of one
or more naturietic peptides of interest would result in an assay
signal by MS that underestimates the concentration of those
naturietic peptides. Deglycosylation can result in an increased
assay signal. Such an increased signal may also be representative
of an increased ability to detect one or more specific forms of the
naturietic peptides of interest. For example, the heterogeneity of
the glycans may result in separation of a single polypeptide into a
plurality of different fractions in a separation method (e.g.,
those based on mass and/or charge). Deglycosylation can result in
coalescence of those different fractions into a single fraction,
thus providing an improved assay signal for that fraction.
[0065] In various embodiments, the increased detection of one or
more naturietic peptides of interest, as compared to performing the
same assaying step in the absence of removing one or more
covalently bound carbohydrate residues from one or more of the
natriuretic peptides of interest, is measured by an assay signal
that increases by at least about 5%, more preferably at least about
10%, still more preferably at least about 20%, even more preferably
at least about 50%, still more preferably at least about 100%, and
most preferably at least about 200% or more.
[0066] In particularly preferred embodiments, the natriuretic
peptides of interest are BNP and/or one or more of its related
fragments. The term "related fragments" is defined hereinafter.
Preferred BNP-related fragments comprise those selected from the
group consisting of pro-BNP (BNP.sub.1-108), NT-proBNP
(BNP.sub.1-76), BNP.sub.3-108, BNP.sub.3-76, and BNP.sub.79-108.
This list is not meant to be limiting.
[0067] In a related aspect, the present invention relates to
methods for selecting and using antibodies that are either
sensitive or insensitive to the presence (or absence) of covalently
bound carbohydrate residues on one or more natriuretic peptides of
interest. Antibodies may be screened for the ability to bind to one
or more glycosylated natriuretic peptides of interest, and that
binding may be compared to the ability to bind to one or more
natriuretic peptides of interest following removal of one or more
covalently bound carbohydrate residues. Those antibodies that
provide substantially identical binding by this measure represent
"insensitive" antibodies. Those antibodies that provide binding by
this measure that is not substantially identical for glycosylated
or deglycosylated forms represent "sensitive" antibodies. Such
antibodies may be selected for use in assay methods for the
detection of one or more natriuretic peptides of interest.
[0068] The term "removal of one or more covalently bound
carbohydrate residues" in this context does not necessarily refer
to the use of enzymatic or non-enzymatic chemical treatments to
remove existing carbohydrate residues from a polypeptide. Instead,
it is meant to encompass any method for generating a polypeptide
lacking one or more covalently bound carbohydrate residues. For
example, solid phase synthesis methods may be used to generate a
polypeptide that is free of all carbohydrate residues for use in
such antibody screening methods. It is preferred that at least
about 50%, more preferably, at least about 60%, still more
preferably at least about 70%, yet more preferably at least about
80%, and most preferably at least about 90% to about 100% of the
carbohydrate residues are removed from one or more, and preferably
all, of the glycosylated natriuretic peptides of interest for use
in the screening methods described herein.
[0069] The term "substantially identical binding" refers to an
antibody that, when used in an assay, provides signals that are
within a factor of about 2 of one another in the screening
comparison described above. A factor of 1 indicates that the
signals are equal; that signals are within a factor of 2 indicates
that one signal is less than or equal to the other signal x 2.
Preferably, antibodies exhibiting substantially identical binding
provide signals that are within a factor of about 1.75, more
preferably within a factor of about 1.5, still more preferably
within a factor of about 1.25, and most preferably within a factor
of about 1.1 to 1.
[0070] Such antibodies may also have "substantially identical
affinity" for one or more glycosylated natriuretic peptides of
interest, as compared to one or more natriuretic peptides of
interest following removal of one or more covalently bound
carbohydrate residues. A factor of 1 indicates that the affinities
are equal; that affinities are within a factor of 2 indicates that
one affinity is less than or equal to the other signal .times.2.
Preferably, antibodies exhibiting substantially identical binding
provide affinities that are within a factor of about 1.75, more
preferably within a factor of about 1.5, still more preferably
within a factor of about 1.25, and most preferably within a factor
of about 1.1 to 1.
[0071] The summary of the invention described above is non-limiting
and other features and advantages of the invention will be apparent
from the following detailed description of the invention, and from
the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0072] FIG. 1 shows a mass spectrum of BNP and its degradation
products in human serum in the absence (panels A and C) and the
presence (panels B and D) of an inhibitor of dipeptidyl
peptidase.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The present invention relates in part to methods for
distinguishing between biologically active (e.g., full length)
natriuretic peptides from biologically inactive forms of the
natriuretic peptides. As described herein, antibodies may be
generated that selectively recognize biologically active
natriuretic peptides, and used in assays that exhibit reduced
inaccuracies caused by the presence of inactive natriuretic peptide
fragments present in a sample.
[0074] The term "natriuretic peptide" as used herein refers to
members of a group of naturally occurring polypeptide hormones that
act in the body to oppose the activity of the renin-angiotensin
system, and their biosynthetic precursors and biologically active
fragments. There are three major human natriuretic peptides: atrial
natriuretic peptide (ANP), which is synthesized in the atria;
brain-type natriuretic peptide (BNP), which is synthesized in the
ventricles; and C-type natriuretic peptide (CNP), which is
synthesized in the brain.
[0075] The term "intact natriuretic peptide" as used herein refers
to the full length pre-pro-natriuretic peptide, full length
pro-natriuretic peptide, full length mature natriuretic peptide,
and/or the full length portions removed during processing of the
pre-pro- or pro-natriuretic peptides during biosynthesis. In the
case of BNP for example, the term "intact natriuretic peptides"
encompasses the full length 32 amino acid mature BNP hormone; the
full length 134-amino acid pre-pro-BNP molecule; the full length
108-amino acid pro-BNP molecule; the full length 76-amino acid
NT-pro BNP molecule, and/or the full length 26-amino acid "pre"
peptide.
[0076] The sequence of the human 108 amino acid BNP precursor
pro-BNP (BNP.sub.1-108) is shown as SEQ ID NO: 1. Mature, full
length BNP (BNP.sub.77-108) is shown underlined:
TABLE-US-00001 HPLGSPGSAS DLETSGLQEQ RNHLQGKLSE LQVEQTSLEP
LQESPRPTGV 50 (SEQ ID NO: 1) WKSREVATEG IRGHRKMVLY TLRAPRSPKM
VQGSGCFGRK MDRISSSSGL 100 GCKVLRRH 108.
[0077] Human BNP.sub.1-108 is synthesized as a larger precursor
pre-pro-BNP having the sequence shown as SEQ ID NO: 2 (with the
"pre" sequence shown in bold):
TABLE-US-00002 MDPQTAPSRA LLLLLFLHLA FLGGRSHPLG SPGSASDLET
SGLQEQRNHL 50 (SEQ ID NO: 2) QGKLSELQVE QTSLEPLQES PRPTGVWKSR
EVATEGIRGH RKMVLYTLRA 100 PRSPKMVQGS GCFGRKMDRI SSSSGLGCKV LRRH
134.
[0078] The sequence of the 126 amino acid human ANP precursor
pro-ANP (ANP.sub.1-126) is shown as SEQ ID NO: 3, with mature, full
length ANP (ANP.sub.99-126) underlined:
TABLE-US-00003 NPMYNAVSNA DLMDFKNLLD HLEEKMPLED EVVPPQVLSD
PNEEAGAALS 50 (SEQ ID NO: 3) PLPEVPPWTG EVSPAQRDGG ALGRGPWDSS
DRSALLKSKL RALLTAPRSL 100 RRSSCFGGRM DRIGAQSGLG CNSFRY 126.
[0079] Human ANP.sub.1-126 is synthesized as a larger precursor
pre-pro-ANP having the sequence shown in SEQ ID NO: 4 (with the
"pre" sequence shown in bold):
TABLE-US-00004 KSSFSTTTVS FLLLLAFQLL GQTRANPMYN AVSNADLMDF
KNLLDHLEEK 50 (SEQ ID NO: 4) MPLEDEVVPP QVLSDPNEEA GAALSPLPEV
PPWTGEVSPA QRDGGALGRG 100 PWDSSDRSAL LKSKLRALLT APRSLRRSSC
FGGRMDRIGA QSGLGCNSFR 150 Y 151.
[0080] The sequence of the 126 amino acid human CNP precursor
pro-CNP (CNP.sub.1-126) is shown as SEQ ID NO: 5, with the full
length mature CNP form CNP-53 (CNP.sub.74-126) shown in italics,
and the full length mature CNP form CNP-22 (CNP.sub.105-126) shown
underlined:
TABLE-US-00005 MHLSQLLACA LLLTLLSLRP SEAKPGAPPK VPRTPPAEEL
AEPQAAGGGQ 50 (SEQ ID NO: 5) KKGDKAPGGG GANLKGDRSR LLRDLRVDTK
SRAAWARLLQ EHPNARKYKG 100 ANKKGLSKGC FCLKLDRIGS MSGLGC 126.
[0081] The term "fragment" as used herein refers to a polypeptide
that comprises at least six contiguous amino acids of a polypeptide
from which the fragment is derived, but is less than the complete
parent polypeptide. Thus, a fragment of pro-BNP (BNP.sub.1-108)
refers to a polypeptide that comprises at least six contiguous
amino acids of BNP.sub.1-108; a fragment of mature BNP refers to a
polypeptide that comprises at least six contiguous amino acids of
BNP.sub.77-108; a fragment of the polypeptide generated by cleavage
of pro-BNP into mature BNP refers to a polypeptide that comprises
at least six contiguous amino acids of BNP.sub.1-76. Similarly, a
fragment of pro-ANP (ANP.sub.1-126) refers to a polypeptide that
comprises at least six contiguous amino acids of ANP.sub.1-126; a
fragment of mature ANP refers to a polypeptide that comprises at
least six contiguous amino acids of ANP.sub.99-126; a fragment of
the polypeptide generated by cleavage of pro-ANP into mature ANP
refers to a polypeptide that comprises at least six contiguous
amino acids of BNP.sub.1-98; and a fragment of pro-CNP
(CNP.sub.1-126) refers to a polypeptide that comprises at least six
contiguous amino acids of CNP.sub.1-126; a fragment of mature CNP
refers to a polypeptide that comprises at least six contiguous
amino acids of CNP.sub.74-126 or CNP.sub.105-126; a fragment of the
polypeptide generated by cleavage of pro-CNP into mature CNP refers
to a polypeptide that comprises at least six contiguous amino acids
of CNP.sub.1-73 or CNP.sub.1-104. In preferred embodiments, a
fragment refers to a polypeptide that comprises at least 10
contiguous amino acids of a polypeptide from which the fragment is
derived; at least 15 contiguous amino acids of a polypeptide from
which the fragment is derived; or at least 20 contiguous amino
acids of a polypeptide from which the fragment is derived.
[0082] The term "related fragment" as used herein refers to one or
more fragments of a particular polypeptide or its biosynthetic
parent that may be detected as a surrogate for the polypeptide
itself or as independent markers. For example, human BNP is derived
by proteolysis of a 108 amino acid precursor molecule, referred to
hereinafter as BNP.sub.1-108. Mature BNP, or "the BNP natriuretic
peptide," or "BNP-32" is a 32 amino acid molecule representing
amino acids 77-108 of this precursor, which may be referred to as
BNP.sub.77-108. The remaining residues 1-76 are referred to
hereinafter as BNP.sub.1-76. BNP.sub.1-108 and BNP.sub.1-76 are
examples of "BNP-related fragments."
[0083] The term "fragment formed by removal of an N-terminal
portion" as used herein in reference to natriuretic peptide
fragments refers to a fragment of an intact natriuretic peptide
formed by removal of one or more amino acids from the amino
terminal end of the intact peptide. In preferred embodiments, such
a fragment is formed by removal of at least 2, 3, 4, 5, 7, 10, 15,
20, or more amino acids from the amino terminal end of the intact
peptide.
[0084] The term "biologically active" as used herein in reference
to natriuretic peptides and fragments thereof refers to a full
length mature natriuretic peptide; or a polypeptide derived from
the full length mature natriuretic peptide or its precursor
molecules that exhibit at least 50% of the vasorelaxation effects
in isolated preconstricted mouse aortic rings exhibited by the full
length mature natriuretic peptide, measured as described in Lopez
et al., J. Biol. Chem. 272: 23064-23068, 1997. Biologically active
natriuretic peptides may include fragments of the full length
mature natriuretic peptide, or precursor forms or fragments
thereof.
[0085] The term "biologically inactive" as used herein in reference
to natriuretic peptide fragments refers to a polypeptide derived
from the full length mature natriuretic peptide or its precursor
that is not "biologically active" as defined above. As used herein,
the term "biologically inactive" does not necessarily refer to a
complete loss of all biological activity. Rather, a "biologically
inactive" natriuretic peptide fragment preferably exhibits less
than 50%, preferably less than 25%, more preferably less than 10%,
and most preferably less than 1%, of one or more biological
functions of the intact natriuretic peptide. This biological
function may be receptor binding, which may be measured as
described in Smith et al., J. Endocrinol. 167: 239-46, 2000, cGMP
production in cultured rat aortic smooth muscle cells, which may be
measured as described in Shimekake et al., FEBS Lett. 309: 185-9,
1992, and/or the vasorelaxation effects in isolated preconstricted
mouse aortic rings exhibited by the full length mature natriuretic
peptide, measured as described in Lopez et al., J. Biol. Chem. 272:
23064-23068, 1997, compared to that exhibited by the full length
mature natriuretic peptide.
[0086] The term "glycosylated" as used herein in regard to
polypeptides refers to polypeptides comprising covalently bound
sugar units, often in the form of glycan chains. The individual
sugar units are referred to herein as "covalently bound
carbohydrate residues." Glycosylation of polypeptides in
eukaryotics occurs principally through glycosidic bonds to an
asparagine side chain ("N-linked"); through glycosidic bonds to
serine or threonine side chains ("O-linked"); or the polypeptide
may be linked to a phosphatidylinositol lipid anchor through a
carbohydrate bridge ("GPI-linked").
[0087] The term "deglycosylation" as used herein refers to methods
for removing one or more covalently bound carbohydrate residues
from polypeptides. While removal of all covalently bound
carbohydrate residues is preferred, a polypeptide is considered to
have been deglycosylated if any covalently bound carbohydrate
residues have been removed. Enzymatic treatments, non-enzymatic
treatments, or a combination of the two may be employed to remove
covalently bound carbohydrate residues from polypeptides. It is
preferred that at least about 50%, more preferably, at least about
60%, still more preferably at least about 70%, yet more preferably
at least about 80%, and most preferably at least about 90% to about
100% of the carbohydrate residues are removed from a
polypeptide.
[0088] As used herein, the term "purified" in reference to
polypeptides does not require absolute purity. Instead, it
represents an indication that the polypeptide(s) of interest
is(are) in a discrete environment in which abundance (on a mass
basis) relative to other proteins is greater than in a biological
sample. By "discrete environment" is meant a single medium, such as
a single solution, a single gel, a single precipitate, etc.
Purified polypeptides may be obtained by a number of methods
including, for example, laboratory synthesis, chromatography,
preparative electrophoresis, centrifugation, precipitation,
affinity purification, etc. One or more "purified" polypeptides of
interest are preferably at least 10% of the protein content of the
discrete environment. One or more "substantially purified"
polypeptides are at least 50% of the protein content of the
discrete environment, more preferably at least 75% of the protein
content of the discrete environment, and most preferably at least
95% of the protein content of the discrete environment. Protein
content is determined using a modification of the method of Lowry
et al., J. Biol. Chem. 193: 265, 1951, described by Hartree, Anal
Biochem 48: 422-427 (1972), using bovine serum albumin as a protein
standard.
[0089] The term "antibody" as used herein refers to a peptide or
polypeptide derived from, modeled after or substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof, capable of specifically binding an antigen or epitope.
See, e.g. Fundamental Immunology, 3rd Edition, W. E. Paul, ed.,
Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods
175:267-273; Yarmush (1992)J. Biochem. Biophys. Methods 25:85-97.
The term antibody includes antigen-binding portions, i.e., "antigen
binding sites," (e.g., fragments, subsequences, complementarity
determining regions (CDRs)) that retain capacity to bind antigen,
including (i) a Fab fragment, a monovalent fragment consisting of
the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent
fragment comprising two Fab fragments linked by a disulfide bridge
at the hinge region; (iii) a Fd fragment consisting of the VH and
CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains
of a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341: 544-546), which consists of a VH domain; and
(vi) an isolated complementarity determining region (CDR). Single
chain antibodies, monoclonal antibodies, polyclonal antibodies, and
antibodies obtained by molecular biological techniques (e.g., by
phage display methods) are also included by reference in the term
"antibody." Preferred antibodies are "Omniclonal" antibodies. By
this is meant a mixture of different antibody molecules selected
from a phage display library, where each antibody specifically
binds to a target antigen with a minimum affinity of
10.sup.9M.sup.-1 to 10.sup.10M.sup.-1.
[0090] The term "specifically binds" is not intended to indicate
that an antibody binds exclusively to its intended target. Rather,
an antibody "specifically binds" if its affinity for its intended
target is about 5-fold greater when compared to its affinity for a
non-target molecule. Preferably the affinity of the antibody will
be at least about 5 fold, preferably 10 fold, more preferably
25-fold, even more preferably 50-fold, and most preferably 100-fold
or more, greater for a target molecule than its affinity for a
non-target molecule. In preferred embodiments, Specific binding
between an antibody or other binding agent and an antigen means a
binding affinity of at least 10.sup.6M.sup.-1. Preferred antibodies
bind with affinities of at least about 10.sup.7 M.sup.-1, and
preferably between about 10.sup.8 M.sup.-1 to about 10.sup.9
M.sup.-1, about 10.sup.9 M.sup.-1 to about 10.sup.10 M.sup.-1, or
about 10.sup.10 M.sup.-1 to about 10.sup.11 M.sup.-1.
[0091] Affinity is calculated as K.sub.d=k.sub.off/k.sub.on
(k.sub.off is the dissociation rate constant, k.sub.on is the
association rate constant and K.sub.d is the equilibrium constant.
Affinity can be determined at equilibrium by measuring the fraction
bound (r) of labeled ligand at various concentrations (c). The data
are graphed using the Scatchard equation: r/c=K(n-r):
[0092] where r=moles of bound ligand/mole of receptor at
equilibrium;
[0093] c=free ligand concentration at equilibrium;
[0094] K=equilibrium association constant; and
[0095] n=number of ligand binding sites per receptor molecule
By graphical analysis, r/c is plotted on the Y-axis versus r on the
X-axis thus producing a Scatchard plot. The affinity is the
negative slope of the line. k.sub.off can be determined by
competing bound labeled ligand with unlabeled excess ligand (see,
e.g., U.S. Pat. No. 6,316,409). The affinity of a targeting agent
for its target molecule is preferably at least about
1.times.10.sup.-6 moles/liter, is more preferably at least about
1.times.10.sup.-7 moles/liter, is even more preferably at least
about 1.times.10.sup.-8 moles/liter, is yet even more preferably at
least about 1.times.10.sup.-9 moles/liter, and is most preferably
at least about 1.times.10.sup.-10 moles/liter. Antibody affinity
measurement by Scatchard analysis is well known in the art. See,
e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and
Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.
[0096] The term "discrete" as used herein refers to areas of a
surface that are non-contiguous. That is, two areas are discrete
from one another if a border that is not part of either area
completely surrounds each of the two areas. The term "independently
addressable" as used herein refers to discrete areas of a surface
from which a specific signal may be obtained. One skilled in the
art will appreciate that antibody zones can also be independent of
each other, but can be in contact with each other on a surface.
[0097] The term "test sample" as used herein refers to a sample in
which the presence or amount of one or more analytes of interest
are unknown and to be determined in an assay, preferably an
immunoassay. Preferably, a test sample is a bodily fluid obtained
for the purpose of diagnosis, prognosis, or evaluation of a
subject, such as a patient. In certain embodiments, such a sample
may be obtained for the purpose of determining the outcome of an
ongoing condition or the effect of a treatment regimen on a
condition. Preferred test samples include blood, serum, plasma,
cerebrospinal fluid, urine and saliva. In addition, one of skill in
the art would realize that some test samples would be more readily
analyzed following a fractionation or purification procedure, for
example, separation of whole blood into serum or plasma components.
Preferred samples may be obtained from bacteria, viruses and
animals, such as dogs and cats. Particularly preferred samples are
obtained from humans. By way of contrast, a "standard sample"
refers to a sample in which the presence or amount of one or more
analytes of interest are known prior to assay for the one or more
analytes.
[0098] The term "disease sample" as used herein refers to a tissue
sample obtained from a subject that has been determined to suffer
from a given disease. Methods for clinical diagnosis are well known
to those of skill in the art. See, e.g., Kelley's Textbook of
Internal Medicine, 4.sup.th Ed., Lippincott Williams & Wilkins,
Philadelphia, Pa., 2000; The Merck Manual of Diagnosis and Therapy,
17.sup.th Ed., Merck Research Laboratories, Whitehouse Station,
N.J., 1999.
[0099] The terms "prolyl-specific dipeptidyl peptidase" or
"prolyl-specific DPP" refer to serine proteases that cleave
dipeptides from the N-terminal of substrate polypeptides, and that
exhibit a preference for proline in the second position (i.e.,
NH2-X-pro-peptide-COOH, where X is an amino acid, and the bond
between pro and the remaining peptide is cleaved). Such proteases
are generally classified under E.C.3.4.14.X, including E.C.3.4.14.5
and 3.4.14.11. DPPs are often classified into types such as DPP-II
and DPP-IV.
[0100] The term "inhibitor" as used herein in reference to
molecules that affect an enzymatic (e.g., proteolytic) activity
does not necessarily refer to a complete loss of all enzymatic
activity. Rather, an "inhibitor" reduces an enzymatic activity by
at least 10%, more preferably at least 25%, even more preferably by
at least 50%, still more preferably by at least 75%, and most
preferably by at least 90%, of the enzymatic activity exhibited in
the absence of the inhibitor. In vitro, the activity of an
inhibitor may be measured by directly measuring enzymatic activity
by methods well known to those of skill in the art. In vivo, the
activity of an inhibitor may also be measured by directly measuring
enzymatic activity on the enzyme substrate, or in the case of a
degradative enzyme, may be measured by determining a time
(T.sub.1/2) in which 1/2 of the substrate is cleared from the body
of a subject (e.g., an experimental animal). In the latter case, an
"inhibitor" increases a T.sub.1/2 by at least 10%, more preferably
at least 25%, even more preferably by at least 50%, still more
preferably by at least 75%, and most preferably by at least 90%,
compared to the T.sub.1/2 exhibited in the absence of the
inhibitor.
[0101] Preferred inhibitors are selective for a particular class of
proteases (e.g., selective for dipeptidyl peptidase or for a
particular subset of dipeptidyl peptidase). An inhibitor is said to
be "selective" for a particular class of protease if it inhibits
that class at least 10-fold more, more preferably at least 100-fold
more, and most preferably at least 1000-fold more, than non-target
proteases. Selective inhibitors of various DPP types are known. For
example, H-Dab-Pip is reportedly be selective (>7,600-fold) for
dipeptidyl peptidase II (DPP II; EC 3.4.14.2) over DPP IV
(IC.sub.50>1 mM) (DPP IV; EC 3.4.14.5). Senten et al., Bioorg.
Med. Chem. Lett. 12: 2825, 2002. Similarly,
1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrol-
idine is reportedly a selective, orally active inhibitor of DPP IV.
Ahren et al., Diabetes Care 25:869-75, 2002.
[0102] The term "about" as used herein refers to +/-10% of a given
number.
[0103] Use of Natriuretic Peptide Fragments as Prognostic and
Diagnostic Markers
[0104] As noted above, increased blood levels of natriuretic
peptides have been found in certain disease states, suggesting a
role in the pathophysiology of those diseases, including stroke,
congestive heart failure (CHF), cardiac ischemia, systemic
hypertension, and acute myocardial infarction. See, e.g., WO
02/089657; WO 02/083913; WO 03/016910; Hunt et al., Biochem.
Biophys. Res. Comm. 214: 1175-83 (1995); Venugopal, J. Clin. Pharm.
Ther. 26: 15-31, 2001; and Kalra et al., Circulation 107: 571-3,
2003; each of which is hereby incorporated in its entirety,
including all tables, figures, and claims. The natriuretic
peptides, alone, collectively, and/or together with additional
proteins, can also serve as disease markers and indicators of
prognosis in various cardiovascular conditions.
[0105] It has been reported that removal of natriuretic peptides
from the circulation involves degradation pathways. Indeed,
inhibitors of neutral endopeptidase, which cleaves natriuretic
peptides under certain circumstances, have been suggested to hold
promise in treatment of certain cardiovascular diseases. See, e.g.,
Trindade and Rouleau, Heart Fail. Monit. 2: 2-7, 2001. However, the
measurement of the natriuretic peptides in clinical samples has
focused generally upon measurement of BNP, ANP, and/or CNP; their
precursor molecules (i.e., pro-BNP, pro-ANP, and pro-CNP); and the
fragments resulting from cleavage of the pro-form to provide the
mature natriuretic peptides, without consideration of the
degradation state of the molecules. It has also been reported that
oxidation of methionine residues in the natriuretic peptides
reduces the biological activity compared to reduced forms. Koyama
et al., Eur. J. Biochem. 203: 425-32. For the purposes described
herein, the methionine-oxidized forms may be considered products of
degradation.
[0106] The present invention describes for the first time that
assays which have not been designed with an understanding of the
degradation pathways of the natriuretic peptides and the products
formed during this degradation, may not accurately measure the
biologically active forms of a particular natriuretic peptide in a
sample. The unintended measurement of both the biologically active
natriuretic peptide(s) of interest and inactive fragments derived
from the natriuretic peptide may result in an overestimation of the
concentration of biologically active natriuretic peptide(s) in a
sample. While described hereinafter mainly with reference to
BNP-related fragments, the skilled artisan will understand that the
general concepts described herein apply equally to ANP- and
CNP-related fragments.
[0107] The failure to consider the activity of the various
natriuretic peptides and their fragments that may be present in a
clinical sample when measuring one or more of the natriuretic
peptides may have serious consequences for the accuracy of any
diagnostic or prognostic method. Consider for example a simple
case, where a sandwich immunoassay is provided for BNP, and a
significant amount (e.g., 50%) of the biologically active BNP that
had been present has now been degraded into an inactive form. An
immunoassay formulated with antibodies that bind a region common to
the biologically active BNP and the inactive fragment(s) will
overestimate the amount of biologically active BNP present in the
sample by 2-fold, potentially resulting in a "false positive"
result. This inaccuracy may be particularly relevant in the case of
severe heart failure, as neutral endopeptidase expression has been
reported to be increased in these patients. Knecht et al., Life
Sci. 71: 2701-12, 2002. This increased expression of the enzyme
believed responsible for natriuretic peptide degradarion could be
expected to increase the inactive fragment pool in these
patients.
[0108] Overestimation of the natriuretic peptide concentration of a
sample may also have serious consequences for patient management.
For example, BNP concentration may be used to determine if therapy
for congestive heart failure is effective (e.g., by monitoring BNP
to see if an elevated level is returning to normal upon treatment).
The same "false positive" BNP result discussed above may lead the
physician to continue, increase, or modify treatment (e.g.,
increase the dosage of diuretic, ACE inhibitor, digoxin,
.beta.-blocker, calcium channel blocker, and/or vasodialtor, or
even consider surgical intervention) because of the false
impression that current therapy is ineffective.
[0109] Similarly, the present invention describes that assays that
have not been designed with an understanding of the glycosylation
state of the natriuretic peptides may likewise not accurately
measure the forms of a particular natriuretic peptide in a sample.
Antibodies are often raised for use in assays through the use of
synthetic peptides or expressed peptides that lack the natural
glycosylation profile seen in vivo. Consider for example a simple
case, where a sandwich immunoassay is provided for BNP, and a
significant amount (e.g., 50%) of the BNP present has glycosylation
that interferes with antibody binding. An immunoassay formulated
with such antibodies will underestimate the amount of BNP present
in the sample by 2-fold, potentially resulting in a "false
negative" result.
[0110] Glycosylation differences amongst the natriuretic peptides
may also result in differences in biological activity, either
through differences in activity at the natriuretic peptide receptor
or through differences in biological half-life due to the
glycosylation state of a particular natriuretic peptide. Thus, the
methods herein may also be used to generate assays that are
specific for certain glycosylation states, again to improve the
accuracy of diagnostic and therapeutic utility of such assays.
[0111] The skilled artisan will understand that the methods
described herein are applicable generally to polypeptides, and the
analysis of the natriuretic peptides described in detail herein is
merely exemplary. Other suitable polypeptides that may be the
subject of similar analysis include angiotensin I, angiotensin II,
vasopressin, calcitonin, calcitonin gene related peptide,
urodilatin, urotensin II, free cardiac troponin I, free cardiac
troponin T, cardiac troponin I in a complex comprising one or both
of troponin T and troponin C, cardiac troponin T in a complex
comprising one or both of troponin I and troponin C, total cardiac
troponin I, total cardiac troponin T, pulmonary surfactant protein
D, D-dimer, annexin V, enolase, creatine kinase, glycogen
phosphorylase, heart-type fatty acid binding protein,
phosphoglyceric acid mutase, S-100, S-100ao,
plasmin-.alpha.2-antiplasmin complex, .beta.-thromboglobulin,
platelet factor 4, fibrinopeptide A, platelet-derived growth
factor, prothrombin fragment 1+2, P-selectin, thrombin-antithrombin
III complex, von Willebrand factor, tissue factor, thrombus
precursor protein, human neutrophil elastase, inducible nitric
oxide synthase, lysophosphatidic acid, malondialdehyde-modified low
density lipoprotein, matrix metalloproteinase-1, matrix
metalloproteinase-2, matrix metalloproteinase-3, matrix
metalloproteinase-9, TIMP1, TIMP2, TIMP3, C-reactive protein,
interleukin-1 .beta., interleukin-1 receptor antagonist,
interleukin-6, tumor necrosis factor .alpha., soluble intercellular
adhesion molecule-1, vascular cell adhesion molecule, monocyte
chemotactic protein-1, caspase-3, human lipocalin-type
prostaglandin D synthase, mast cell tryptase, eosinophil cationic
protein, KL-6, procalcitonin, haptoglobin, s-CD40 ligand, S-FAS
ligand, alpha 2 actin, basic calponin 1, CSRP2 elastin, LTBP4,
smooth muscle myosin, smooth muscle myosin heavy chain, transgelin,
aldosterone, angiotensin III, bradykinin, endothelin 1, endothelin
2, endothelin 3, renin, APO B48, pancreatic elastase 1, pancreatic
lipase, sPLA2, trypsinogen activation peptide, alpha enolase,
LAMP3, phospholipase D, PLA2G5, protein D, SFTPC, defensin HBD1,
defensin HBD2, CXCL-1, CXCL-2, CXCL-3, CCL2, CCL3, CCL4, CCL8,
procalcitonin, protein C, serum amyloid A, s-glutathione, s-TNF
P55, s-TNF P75, TAFI, TGF beta, MMP-11, brain fatty acid binding
protein, CA11, CABP1, CACNA1A, CBLN1, CHN2, cleaved Tau, CRHR1,
DRPLA, EGF, GPM6B, GPR7, GPR8, GRIN2C, GRM7, HAPIP, HIF 1 alpha,
HIP2 KCNK4, KCNK9, KCNQ5, MAPK10, n-acetyl aspartate, NEUROD2,
NRG2, PACE4, phosphoglycerate mutase, PKC gamma, prostaglandin E2,
PTEN, PTPRZ1, RGS9, SCA7, secretagogin, SLC1A3, SORL1, SREB3, STAC,
STX1A, STXBP1, BDNF, cystatin C, neurokinin A, substance P,
interleukin-1, interleukin-11, interleukin-13, interleukin-18,
interleukin-4, and interleukin-10.
[0112] Glycosylation of Natriuretic Peptides
[0113] Glycosylated polypeptides typically comprise N-linked sugars
attached to the amino group of one or more asparagine residues;
O-linked sugars attached to the hydroxyl group of one or more
serine and/or threonine residues; or a combination of N- and
O-linked sugars. The present invention demonstrates for the first
time that natriuretic peptides are glycosylated. Furthermore, the
present invention demonstrates that glycosylation can significantly
affect the ability of certain methods of detecting natriuretic
peptides in samples.
[0114] Several approaches may be used to obviate the potential
difficulties presented by glycosylation to a detection scheme.
First, one may use chemical or enzymatic treatments to remove
carbohydrate residues from the polypeptides, thereby shifting one
or more of the naturietic peptides of interest to a "detectable"
state if the presence of glycosylation disrupting accurate
detection. Second, one may carefully select antibodies that bind to
one or more regions of the naturietic peptides of interest that are
not subject to interference by glycosylation to provide antibodies
that are "insensitive" to a particular glycosylation state. Third,
one may carefully select antibodies that bind to one or more
regions of the naturietic peptides of interest that are
glycosylated, but that exhibit reduced binding in the
deglycosylated state, to provide antibodies that are "sensitive" to
a particular glycosylation state. Fourth, one may carefully select
antibodies that bind to one or more regions of the naturietic
peptides of interest that are glycosylated, but that exhibit
increased binding in the deglycosylated state, to provide
antibodies that are "sensitive" to a particular glycosylation
state. One may also combine these approaches as necessary or
desired.
[0115] Effective enzymatic methods for removing N- and O-linked
carbohydrate residues are well known in the art, using enzymes such
as N-glycanase (also known as N-glycosidase), endoglycosidase H,
endoglycosidase A, O-glycanase (also known as
endo-.alpha.-N-acetylgalactosaminidase),
.alpha.2-(3,6,8,9)-neuriminidase, .beta.(1,4)-galactosidase,
N-acetylglucosaminidase, endoglycosidase F.sub.1, endoglycosidase
F.sub.2, and/or endoglycosidase F.sub.3. This list is not meant to
be limiting. Such enzymatic methods of sugar removal from peptides
may be used on native (non-denatured) peptides. In such enzymatic
methods, however, denaturation of the glycopeptide may be employed,
often with an increased rate of deglycosylation. Common
denaturation conditions comprise the addition of about 0.01% to
about 1% sodium dodecyl sulfate ("SDS"), and optionally about 5 mM
to about 500 mM .beta.-mercaptoethanol, in a buffer solution at
about neutral pH (i.e., between about pH 6.5 and about pH 8). Such
methods may further comprise from about 0.2% to about 2% NP-40,
which can serve to stabilize some deglycosylation enzymes.
Increased temperature (e.g., about 37.degree. C. for from about 0.5
hours to about 48 hours) may also be employed together with such
denaturation conditions.
[0116] In the case of non-enzymatic chemical treatments for removal
of covalently bound carbohydrate residues from peptides, hydrazine
hydrolysis has been found to be effective in the release of
unreduced O- and N-linked oligosaccharides. Selective and
sequential release of oligosaccharides can be accomplished by
initial mild hydrazinolysis of the O-linked oligosaccharides at
about 60.degree. C. followed by N-linked oligosaccharides at about
95.degree. C. See, e.g., Patel and Rarekh, Meth. Enzymol. 230,
58-66, 1994. Such treatment may result in destruction of the
polypeptide however. Alkaline-.beta.-elimination of O-linked
oligosaccharides, which utilizes alkaline sodium borohydride in a
mild base environment, may be preferred. See, e.g., Glycobiology: A
Practical Approach, Fukuda, M. and Kobata, A. (Eds), pp. 291-328,
IRL/Oxford Univ. Press, Oxford, 1993. In addition,
trifluoromethanesulfonic acid hydrolysis may be employed. This
method typically leaves an intact polypeptide, but results in
destruction of the glycan, as glycosyl linkages between sugars are
sensitive to cleavage by trifluoromethanesulfonic acid, but peptide
bonds are stable to even prolonged treatment. See, e.g., Edge,
Biochem. J. 376: 339-50, 2003.
[0117] Importantly, changes in mass observed in peptides following
such enzymatic or trifluoromethanesulfonic acid treatment can be
ascribed to removal of sugar residues, as post-translational
modifications other than glycosylation are believed to be stable to
such treatments. This can allow for better understanding of the
relative contribution of carbohydrates and glycosylation sites to
the antigenic epitopes on the polypeptides of interest.
Deglycoylation can also allow better understanding of differences
in polypeptide mass (e.g., the mass of the natriuretic peptides of
interest and fragments thereof present in a sample, which can be
related by methods well known to those of skill in the art to the
sequence), as the removal of sugar residues removes any doubt as to
whether differences in mass observed may be due to differences in
sugar content rather than amino acid content.
[0118] Selection of Antibodies
[0119] The generation and selection of antibodies that
preferentially recognize intact natriuretic peptides fragments
and/or are sensitive or insensitive to glycosylation state may be
accomplished several ways. For example, one way is to purify
fragments or to synthesize the fragments of interest using, e.g.,
solid phase peptide synthesis methods well known in the art. See,
e.g., Guide to Protein Purification, Murray P. Deutcher, ed., Meth.
Enzymol. Vol 182 (1990); Solid Phase Peptide Synthesis, Greg B.
Fields ed., Meth. Enzymol.Vol 289 (1997); Kiso et al., Chem. Pharm.
Bull. (Tokyo) 38: 1192-99, 1990; Mostafavi et al., Biomed. Pept.
Proteins Nucleic Acids 1: 255-60, 1995; Fujiwara et al., Chem.
Pharm. Bull. (Tokyo) 44: 1326-31, 1996. The selected polypeptides
may then be injected, for example, into mice or rabbits, to
generate polyclonal or monoclonal antibodies. One skilled in the
art will recognize that many procedures are available for the
production of antibodies, for example, as described in Antibodies,
A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor
Laboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art
will also appreciate that binding fragments or Fab fragments which
mimic antibodies can also be prepared from genetic information by
various procedures (Antibody Engineering: A Practical Approach
(Borrebaeck, C., ed.), 1995, Oxford University Press, Oxford; J.
Immunol. 149, 3914-3920 (1992)).
[0120] In addition, numerous publications have reported the use of
phage display technology to produce and screen libraries of
polypeptides for binding to a selected target. See, e.g, Cwirla et
al., Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et al.,
Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88,
1990; and Ladner et al., U.S. Pat. No. 5,571,698. A basic concept
of phage display methods is the establishment of a physical
association between DNA encoding a polypeptide to be screened and
the polypeptide. This physical association is provided by the phage
particle, which displays a polypeptide as part of a capsid
enclosing the phage genome which encodes the polypeptide. The
establishment of a physical association between polypeptides and
their genetic material allows simultaneous mass screening of very
large numbers of phage bearing different polypeptides. Phage
displaying a polypeptide with affinity to a target bind to the
target and these phage are enriched by affinity screening to the
target. The identity of polypeptides displayed from these phage can
be determined from their respective genomes. Using these methods a
polypeptide identified as having a binding affinity for a desired
target can then be synthesized in bulk by conventional means. See,
e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in its
entirety, including all tables, figures, and claims.
[0121] The antibodies that are generated by these methods may then
be selected by first screening for affinity and specificity with
the purified intact natriuretic peptide of interest and, if
required, comparing the results to the affinity and specificity of
the antibodies with natriuretic fragments that are desired to be
excluded from binding. The screening procedure can involve
immobilization of the purified natriuretic fragments in separate
wells of microtiter plates. The solution containing a potential
antibody or groups of antibodies is then placed into the respective
microtiter wells and incubated for about 30 min to 2 h. The
microtiter wells are then washed and a labeled secondary antibody
(for example, an anti-mouse antibody conjugated to alkaline
phosphatase if the raised antibodies are mouse antibodies) is added
to the wells and incubated for about 30 min and then washed.
Substrate is added to the wells and a color reaction will appear
where antibody to the immobilized natriuretic peptide(s) and
fragment(s) are present. A similar approach may be used to screen
glycosylation-insensitive antibodies. In this case, screening may
take place using purified natriuretic fragments containing and
lacking one or more carbohydrate residues.
[0122] The antibodies so identified may then be further analyzed
for affinity and specificity to the natriuretic peptide(s) of
interest in the assay design selected. In the development of
immunoassays for a target protein, the purified target protein acts
as a standard with which to judge the sensitivity and specificity
of the immunoassay using the antibodies that have been selected.
Because the binding affinity of various antibodies may differ;
certain antibody pairs (e.g., in sandwich assays) may interfere
with one another sterically, etc., assay performance of an antibody
may be a more important measure than absolute affinity and
specificity of an antibody.
[0123] Those skilled in the art will recognize that many approaches
can be taken in producing antibodies or binding fragments and
screening and selecting for affinity and specificity for the
various natriuretic peptides, but these approaches do not change
the scope of the invention.
[0124] Use of Natriuretic Peptides in Marker Panels
[0125] Methods and systems for the identification of one or more
markers for the diagnosis, and in particular for the differential
diagnosis, of disease have been described previously. Suitable
methods for identifying markers useful for the diagnosis of disease
states are described in detail in U.S. patent application Ser. No.
10/331,127, entitled METHOD AND SYSTEM FOR DISEASE DETECTION USING
MARKER COMBINATIONS (attorney docket no. 071949-6802), filed Dec.
27, 2002, which is hereby incorporated by reference in its
entirety, including all tables, figures, and claims. One skilled in
the art will also recognize that univariate analysis of markers can
be performed and the data from the univariate analyses of multiple
markers can be combined to form panels of markers to differentiate
different disease conditions.
[0126] In developing a panel of markers useful in diagnosis, data
for a number of potential markers may be obtained from a group of
subjects by testing for the presence or level of certain markers.
The group of subjects is divided into two sets, and preferably the
first set and the second set each have an approximately equal
number of subjects. The first set includes subjects who have been
confirmed as having a disease or, more generally, being in a first
condition state. For example, this first set of patients may be
those that have recently had a disease incidence, or may be those
having a specific type of disease. The confirmation of the
condition state may be made through a more rigorous and/or
expensive testing such as MRI or CT. Hereinafter, subjects in this
first set will be referred to as "diseased".
[0127] The second set of subjects is simply those who do not fall
within the first set. Subjects in this second set may be
"non-diseased;" that is, normal subjects. Alternatively, subjects
in this second set may be selected to exhibit one symptom or a
constellation of symptoms that mimic those symptoms exhibited by
the "diseased" subjects. In still another alternative, this second
set may represent those at a different time point from disease
incidence.
[0128] The data obtained from subjects in these sets includes
levels of a plurality of markers, including for purposes of the
present invention, one or more fragments of natriuretic peptides
either measured individually or as a group. Preferably, data for
the same set of markers is available for each patient. This set of
markers may include all candidate markers which may be suspected as
being relevant to the detection of a particular disease or
condition. Actual known relevance is not required. Embodiments of
the methods and systems described herein may be used to determine
which of the candidate markers are most relevant to the diagnosis
of the disease or condition. The levels of each marker in the two
sets of subjects may be distributed across a broad range, e.g., as
a Gaussian distribution. However, no distribution fit is
required.
[0129] A marker often is incapable of definitively identifying a
patient as either diseased or non-diseased. For example, if a
patient is measured as having a marker level that falls within the
overlapping region, the results of the test will be useless in
diagnosing the patient. An artificial cutoff may be used to
distinguish between a positive and a negative test result for the
detection of the disease or condition. Regardless of where the
cutoff is selected, the effectiveness of the single marker as a
diagnosis tool is unaffected. Changing the cutoff merely trades off
between the number of false positives and the number of false
negatives resulting from the use of the single marker. The
effectiveness of a test having such an overlap is often expressed
using a ROC (Receiver Operating Characteristic) curve. ROC curves
are well known to those skilled in the art.
[0130] The horizontal axis of the ROC curve represents
(1-specificity), which increases with the rate of false positives.
The vertical axis of the curve represents sensitivity, which
increases with the rate of true positives. Thus, for a particular
cutoff selected, the value of (1-specificity) may be determined,
and a corresponding sensitivity may be obtained. The area under the
ROC curve is a measure of the probability that the measured marker
level will allow correct identification of a disease or condition.
Thus, the area under the ROC curve can be used to determine the
effectiveness of the test.
[0131] As discussed above, the measurement of the level of a single
marker may have limited usefulness. The measurement of additional
markers provides additional information, but the difficulty lies in
properly combining the levels of two potentially unrelated
measurements. In the methods and systems according to embodiments
of the present invention, data relating to levels of various
markers for the sets of diseased and non-diseased patients may be
used to develop a panel of markers to provide a useful panel
response. The data may be provided in a database such as Microsoft
Access, Oracle, other SQL databases or simply in a data file. The
database or data file may contain, for example, a patient
identifier such as a name or number, the levels of the various
markers present, and whether the patient is diseased or
non-diseased.
[0132] Next, an artificial cutoff region may be initially selected
for each marker. The location of the cutoff region may initially be
selected at any point, but the selection may affect the
optimization process described below. In this regard, selection
near a suspected optimal location may facilitate faster convergence
of the optimizer. In a preferred method, the cutoff region is
initially centered about the center of the overlap region of the
two sets of patients. In one embodiment, the cutoff region may
simply be a cutoff point. In other embodiments, the cutoff region
may have a length of greater than zero. In this regard, the cutoff
region may be defined by a center value and a magnitude of length.
In practice, the initial selection of the limits of the cutoff
region may be determined according to a pre-selected percentile of
each set of subjects. For example, a point above which a
pre-selected percentile of diseased patients are measured may be
used as the right (upper) end of the cutoff range.
[0133] Each marker value for each patient may then be mapped to an
indicator. The indicator is assigned one value below the cutoff
region and another value above the cutoff region. For example, if a
marker generally has a lower value for non-diseased patients and a
higher value for diseased patients, a zero indicator will be
assigned to a low value for a particular marker, indicating a
potentially low likelihood of a positive diagnosis. In other
embodiments, the indicator may be calculated based on a polynomial.
The coefficients of the polynomial may be determined based on the
distributions of the marker values among the diseased and
non-diseased subjects.
[0134] The relative importance of the various markers may be
indicated by a weighting factor. The weighting factor may initially
be assigned as a coefficient for each marker. As with the cutoff
region, the initial selection of the weighting factor may be
selected at any acceptable value, but the selection may affect the
optimization process. In this regard, selection near a suspected
optimal location may facilitate faster convergence of the
optimizer. In a preferred method, acceptable weighting coefficients
may range between zero and one, and an initial weighting
coefficient for each marker may be assigned as 0.5. In a preferred
embodiment, the initial weighting coefficient for each marker may
be associated with the effectiveness of that marker by itself. For
example, a ROC curve may be generated for the single marker, and
the area under the ROC curve may be used as the initial weighting
coefficient for that marker.
[0135] Next, a panel response may be calculated for each subject in
each of the two sets. The panel response is a function of the
indicators to which each marker level is mapped and the weighting
coefficients for each marker. In a preferred embodiment, the panel
response (R) for a each subject (j) is expressed as:
R.sub.j=w.sub.iI.sub.i,j,
where i is the marker index, j is the subject index, w.sub.i is the
weighting coefficient for marker i, I is the indicator value to
which the marker level for marker i is mapped for subject j, and
.SIGMA. is the summation over all candidate markers i.
[0136] One advantage of using an indicator value rather than the
marker value is that an extraordinarily high or low marker levels
do not change the probability of a diagnosis of diseased or
non-diseased for that particular marker. Typically, a marker value
above a certain level generally indicates a certain condition
state. Marker values above that level indicate the condition state
with the same certainty. Thus, an extraordinarily high marker value
may not indicate an extraordinarily high probability of that
condition state. The use of an indicator which is constant on one
side of the cutoff region eliminates this concern.
[0137] The panel response may also be a general function of several
parameters including the marker levels and other factors including,
for example, race and gender of the patient. Other factors
contributing to the panel response may include the slope of the
value of a particular marker over time. For example, a patient may
be measured when first arriving at the hospital for a particular
marker. The same marker may be measured again an hour later, and
the level of change may be reflected in the panel response.
Further, additional markers may be derived from other markers and
may contribute to the value of the panel response. For example, the
ratio of values of two markers may be a factor in calculating the
panel response.
[0138] Having obtained panel responses for each subject in each set
of subjects, the distribution of the panel responses for each set
may now be analyzed. An objective function may be defined to
facilitate the selection of an effective panel. The objective
function should generally be indicative of the effectiveness of the
panel, as may be expressed by, for example, overlap of the panel
responses of the diseased set of subjects and the panel responses
of the non-diseased set of subjects. In this manner, the objective
function may be optimized to maximize the effectiveness of the
panel by, for example, minimizing the overlap.
[0139] In a preferred embodiment, the ROC curve representing the
panel responses of the two sets of subjects may be used to define
the objective function. For example, the objective function may
reflect the area under the ROC curve. By maximizing the area under
the curve, one may maximize the effectiveness of the panel of
markers. In other embodiments, other features of the ROC curve may
be used to define the objective function. For example, the point at
which the slope of the ROC curve is equal to one may be a useful
feature. In other embodiments, the point at which the product of
sensitivity and specificity is a maximum, sometimes referred to as
the "knee," may be used. In an embodiment, the sensitivity at the
knee may be maximized. In further embodiments, the sensitivity at a
predetermined specificity level may be used to define the objective
function. Other embodiments may use the specificity at a
predetermined sensitivity level may be used. In still other
embodiments, combinations of two or more of these ROC-curve
features may be used.
[0140] It is possible that one of the markers in the panel is
specific to the disease or condition being diagnosed. When such
markers are present at above or below a certain threshold, the
panel response may be set to return a "positive" test result. When
the threshold is not satisfied, however, the levels of the marker
may nevertheless be used as possible contributors to the objective
function.
[0141] An optimization algorithm may be used to maximize or
minimize the objective function. Optimization algorithms are
well-known to those skilled in the art and include several commonly
available minimizing or maximizing functions including the Simplex
method and other constrained optimization techniques. It is
understood by those skilled in the art that some minimization
functions are better than others at searching for global minimums,
rather than local minimums. In the optimization process, the
location and size of the cutoff region for each marker may be
allowed to vary to provide at least two degrees of freedom per
marker. Such variable parameters are referred to herein as
independent variables. In a preferred embodiment, the weighting
coefficient for each marker is also allowed to vary across
iterations of the optimization algorithm. In various embodiments,
any permutation of these parameters may be used as independent
variables.
[0142] In addition to the above-described parameters, the sense of
each marker may also be used as an independent variable. For
example, in many cases, it may not be known whether a higher level
for a certain marker is generally indicative of a diseased state or
a non-diseased state. In such a case, it may be useful to allow the
optimization process to search on both sides. In practice, this may
be implemented in several ways. For example, in one embodiment, the
sense may be a truly separate independent variable which may be
flipped between positive and negative by the optimization process.
Alternatively, the sense may be implemented by allowing the
weighting coefficient to be negative.
[0143] The optimization algorithm may be provided with certain
constraints as well. For example, the resulting ROC curve may be
constrained to provide an area-under-curve of greater than a
particular value. ROC curves having an area under the curve of 0.5
indicate complete randomness, while an area under the curve of 1.0
reflects perfect separation of the two sets. Thus, a minimum
acceptable value, such as 0.75, may be used as a constraint,
particularly if the objective function does not incorporate the
area under the curve. Other constraints may include limitations on
the weighting coefficients of particular markers. Additional
constraints may limit the sum of all the weighting coefficients to
a particular value, such as 1.0.
[0144] The iterations of the optimization algorithm generally vary
the independent parameters to satisfy the constraints while
minimizing or maximizing the objective function. The number of
iterations may be limited in the optimization process. Further, the
optimization process may be terminated when the difference in the
objective function between two consecutive iterations is below a
predetermined threshold, thereby indicating that the optimization
algorithm has reached a region of a local minimum or a maximum.
[0145] Thus, the optimization process may provide a panel of
markers including weighting coefficients for each marker and cutoff
regions for the mapping of marker values to indicators. In order to
develop lower-cost panels which require the measurement of fewer
marker levels, certain markers may be eliminated from the panel. In
this regard, the effective contribution of each marker in the panel
may be determined to identify the relative importance of the
markers. In one embodiment, the weighting coefficients resulting
from the optimization process may be used to determine the relative
importance of each marker. The markers with the lowest coefficients
may be eliminated.
[0146] In certain cases, the lower weighting coefficients may not
be indicative of a low importance. Similarly, a higher weighting
coefficient may not be indicative of a high importance. For
example, the optimization process may result in a high coefficient
if the associated marker is irrelevant to the diagnosis. In this
instance, there may not be any advantage that will drive the
coefficient lower. Varying this coefficient may not affect the
value of the objective function.
[0147] Use of Natriuretic Peptides for Determining a Treatment
Regimen
[0148] A useful diagnostic or prognostic indicator such as the
natriuretic peptides can help clinicians select between alternative
therapeutic regimens. For example, patients with elevation in
cardiac troponin T or I following an acute coronary syndrome appear
to derive specific benefit from an early aggressive strategy that
includes potent antiplatelet and antithrombotic therapy, and early
revascularization. Hamm et al., N. Engl. J. Med. 340: 1623-9
(1999); Morrow et al., J. Am. Coll. Cardiol. 36: 1812-7 (2000);
Cannon et al., Am. J. Cardiol. 82: 731-6 (1998). Additionally,
patients with elevation in C-reactive protein following myocardial
infarction appear to derive particular benefit from HMG-CoA
Reductase Inhibitor therapy. Ridker et al., Circulation 98: 839-44
(1998). Among patients with congestive heart failure, pilot studies
suggest that ACE inhibitors may reduce BNP levels in a dose
dependent manner. Van Veldhuisen et al., J. Am. Coll. Cardiol. 32:
1811-8 (1998).
[0149] Similarly, "tailoring" diuretic and vasodilator therapy
based on the level of the biologically active natriuretic peptides
may improve outcomes. See, e.g., Troughton et al., Lancet 355:
1126-30 (2000). Finally, in a single pilot study of 16 patients
found that randomization to an ACE inhibitor rather than placebo
following Q-wave MI was associated with reduced BNP levels over the
subsequent 6-month period. Motwani et al., Lancet 341: 1109-13
(1993). Because BNP is a counter-regulatory hormone with beneficial
cardiac and renal effects, it is likely that a change in BNP
concentration reflects improved ventricular function and reduced
ventricular wall stress. A recent article demonstrates the
correlation of NT pro-BNP and BNP assays (Fischer et al., Clin.
Chem. 47: 591-594 (2001). It is a further objective of this
invention that the concentration of natriuretic peptides, either
individually or considered in groups of markers, can be used to
guide diuretic and vasodilator therapy to improve patient outcome.
Additionally, the measurement of natriuretic peptides, either
individually or considered in groups of markers, for use as a
prognostic indicator for patients is within the scope of the
present invention.
[0150] Recent studies in patients hospitalized with congestive
heart failure suggest that serial BNP measurements may provide
incremental prognostic information as compared to a single
measurement; that is, assays can demonstrate an improving prognosis
when BNP falls after therapy than when it remains persistently
elevated. Cheng et al., J. Am. Coll. Cardiol. 37: 386-91 (2001).
Thus, serial measurements of natriuretic peptides according to the
present invention may increase the prognostic and/or diagnostic
value of a marker in patients, and is thus within the scope of the
present invention.
[0151] Assay Measurement Strategies
[0152] Numerous methods and devices are well known to the skilled
artisan for the detection and analysis of polypeptides or proteins
in test samples. In preferred embodiments, immunoassay devices and
methods are often used. See, e.g., U.S. Pat. Nos. 6,143,576;
6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615;
5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and
5,480,792, each of which is hereby incorporated by reference in its
entirety, including all tables, figures and claims. These devices
and methods can utilize labeled molecules in various sandwich,
competitive, or non-competitive assay formats, to generate a signal
that is related to the presence or amount of an analyte of
interest. Additionally, certain methods and devices, such as
biosensors and optical immunoassays, may be employed to determine
the presence or amount of analytes without the need for a labeled
molecule. See, e.g., U.S. Pat. Nos. 5,631,171; and 5,955,377, each
of which is hereby incorporated by reference in its entirety,
including all tables, figures and claims. One skilled in the art
also recognizes that robotic instrumentation including but not
limited to Beckman Access, Abbott AxSym, Roche ElecSys, Dade
Behring Stratus systems are among the immunoassay analyzers that
are capable of performing the immunoassays taught herein. Specific
immunological binding of the antibody to the marker can be detected
directly or indirectly. Direct labels include fluorescent or
luminescent tags, metals, dyes, radionuclides, and the like,
attached to the antibody. Indirect labels include various enzymes
well known in the art, such as alkaline phosphatase, horseradish
peroxidase and the like.
[0153] The use of immobilized antibodies specific for the one or
more polypeptides is also contemplated by the present invention.
The antibodies could be immobilized onto a variety of solid
supports, such as magnetic or chromatographic matrix particles, the
surface of an assay place (such as microtiter wells), pieces of a
solid substrate material or membrane (such as plastic, nylon,
paper), and the like. An assay strip could be prepared by coating
the antibody or a plurality of antibodies in an array on solid
support. This strip could then be dipped into the test sample and
then processed quickly through washes and detection steps to
generate a measurable signal, such as a colored spot.
[0154] The analysis of a plurality of polypeptides may be carried
out separately or simultaneously with one test sample. For separate
or sequential assay, suitable apparatuses include clinical
laboratory analyzers such as the ElecSys (Roche), the AxSym
(Abbott), the Access (Beckman), the ADVIA.RTM. CENTAUR.RTM. (Bayer)
immunoassay systems, the NICHOLS ADVANTAGE.RTM. (Nichols Institute)
immunoassay system, etc. Preferred apparatuses or protein chips
perform simultaneous assays of a plurality of polypeptides on a
single surface. Particularly useful physical formats comprise
surfaces having a plurality of discrete, addressable locations for
the detection of a plurality of different analytes. Such formats
include protein microarrays, or "protein chips" (see, e.g., Ng and
Ilag, J. Cell Mol. Med. 6: 329-340 (2002)) and certain capillary
devices (see, e.g., U.S. Pat. No. 6,019,944). In these embodiments,
each discrete surface location may comprise antibodies to
immobilize one or more analyte(s) (e.g., one or more polypeptides
of the invention) for detection at each location. Surfaces may
alternatively comprise one or more discrete particles (e.g.,
microparticles or nanoparticles) immobilized at discrete locations
of a surface, where the microparticles comprise antibodies to
immobilize one analyte (e.g., one or more polypeptides of the
invention) for detection.
[0155] In addition, one skilled in the art would recognize the
value of testing multiple samples (for example, at successive time
points) from the same individual. Such testing of serial samples
will allow the identification of changes in polypeptide levels over
time. Increases or decreases in polypeptide levels, as well as the
absence of change in such levels, would provide useful information
about the disease status that includes, but is not limited to
identifying the approximate time from onset of the event, the
presence and amount of salvageable tissue, the appropriateness of
drug therapies, the effectiveness of various therapies as indicated
by reperfusion or resolution of symptoms, differentiation of the
various types of disease having similar symptoms, identification of
the severity of the event, identification of the disease severity,
and identification of the patient's outcome, including risk of
future events.
[0156] A panel consisting of the polypeptides referenced above, and
optionally including other protein markers useful in diagnosis,
prognosis, or differentiation of disease, may be constructed to
provide relevant information related to differential diagnosis.
Such a panel may be constructed to detect 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15, 20, or more or individual analytes, including one or
more polypeptides of the present invention. The analysis of a
single analyte or subsets of analytes could be carried out by one
skilled in the art to optimize clinical sensitivity or specificity
in various clinical settings. These include, but are not limited to
ambulatory, urgent care, critical care, intensive care, monitoring
unit, inpatient, outpatient, physician office, medical clinic, and
health screening settings. Furthermore, one skilled in the art can
use a single analyte or a subset of analytes in combination with an
adjustment of the diagnostic threshold in each of the
aforementioned settings to optimize clinical sensitivity and
specificity. The clinical sensitivity of an assay is defined as the
percentage of those with the disease that the assay correctly
predicts, and the specificity of an assay is defined as the
percentage of those without the disease that the assay correctly
predicts (Tietz Textbook of Clinical Chemistry, 2.sup.nd edition,
Carl Burtis and Edward Ashwood eds., W.B. Saunders and Company, p.
496).
[0157] The analysis of analytes could be carried out in a variety
of physical formats as well. For example, the use of microtiter
plates or automation could be used to facilitate the processing of
large numbers of test samples. Alternatively, single sample formats
could be developed to facilitate immediate treatment and diagnosis
in a timely fashion, for example, in ambulatory transport or
emergency room settings.
[0158] As discussed above, samples may continue to degrade the
natriuretic peptides or fragments thereof, even once the sample is
obtained. Thus, it may be advantageous to add one or more protease
inhibitors to samples prior to assay. Numerous protease inhibitors
are known to those of skill in the art, and exemplary inhibitors
may be found in, e.g., The Complete Guide for Protease Inhibition,
Roche Molecular Biochemicals, updated Jun. 3, 1999 at
http://www.roche-applied-science.com/fst/products.htm?/prod_inf/manuals/p-
rotease/prot_toc.htm, and European Patent Application 03013792.1
(published as EP 1 378 242 A1), each of which is hereby
incorporated in its entirety. Because various metalloproteases and
calcium-dependent proteases are known to exist in blood-derived
samples, chelators such as EGTA and/or EDTA, also act as protease
inhibitors. In addition, or in the alternative, inhibitors of
neutral endopeptidase and/or DPPs may be used.
[0159] Inhibition of Natriuretic Peptide Degradation by
Prolyl-Specific DPPs
[0160] The neurohumoral regulatory system of which natriuretic
peptides are a part represents a complex system of cardiovascular
regulation. Diseases such as congestive heart failure are, in
essence, fatal diseases for which life may be prolonged, but the
underlying disease never cured. Thus, there remains a need for
novel therapeutic approaches to the management of the underlying
diseases, and multiple points in this complex system are seen as
important targets by clinicians. The clinical success of
angiotensin converting enzyme ("ACE") inhibitors in disease
management has led to a search for additional approaches that
indirectly affect the course of cardiovascular disease by affecting
enzymes that act on vasoactive hormones.
[0161] In the case of the natriuretic hormones, increasing hormone
levels have been found to have therapeutic potential in patients.
See, e.g., Tsekoura et al., Hellenic J. Cardiol. 44: 266-70, 2003.
Neutral endopeptidase ("NEP") is believed to be a key degradation
mediator. Not surprisingly, inhibitors of NEP have found use in
treating patients with diseases such as hypertension,
atherosclerosis, and heart failure. See, e.g., Corti et
al.,Circulation 104: 1856-62, 2001. Combination treatment with both
BNP and NEP inhibitors has been reported to produce a synergistic
effect on cardiac output, reduced vascular resistance, and
unloading of the heart. Chen et al., Circulation 105: 999-1003,
2002. Targeting NEP may suffer from the limitation, however, that
NEP metabolizes a broad range of biologically active peptides. See,
e.g., Walter et al., Curr. Opin. Nephrol. Hypertens. 6: 468-73,
1997.
[0162] The present invention describes a novel approach to
treatment of cardiovascular disease, particularly heart failure.
Several natriuretic peptides, including human forms of pro-BNP,
mature BNP, and pro-ANP comprise a penultimate proline residue,
rendering the peptides suitable substrates for prolyl-specific
dipeptidyl dipeptidases ("DPPs"). Inhibitors of DPP have been
described as having utility in the management of diabetes, mediated
by the inhibition of glucose-dependent insulinotropic polypeptide
degradation by DPP IV. See, e.g., Gault et al., Biochem. Biophys.
Res. Commun. 22: 207-13, 2003. However, their use in treatment of
cardiovascular disease has not previously been reported.
[0163] Methods for preparing and identifying selective DPP
inhibitors are well known in the art. DPP inhibitors include the
dipeptide analogues Xaa-boroPro, including Pro-boroPro,
Ala-boroPro, Val-boroPro, and Lys-boroPro, and dab-pip. See, e.g.,
Senten et al., Bioorg. Med. Chem. Lett. 12: 2825-28, 2002; Jones et
al., Blood, prepublished online May 8, 2003; DOI 10.1182.
Combinatorial chemistry methods have been used to rapidly
synthesize and screen numerous additional dipeptide analogue
inhibitors of DPP. See, e.g., Leiting et al., Biochem. J. 371:
525-32, 2003; Sedo et al., Physiol. Res. 52: 367-72, 2003;
Villhauer et al., J. Med. Chem. 46: 2774-89, 2003; Senten et al., J
Comb. Chem. 5: 336-44, 2003; and U.S. Pat. Nos. 5,602,102;
6,573,287, 6,548,481, 6,432,969, and 6,355,614. The compounds
described in these publications may be used as lead compounds in
identifying additional DPP inhibitors for use in the methods
described herein. A variety of techniques are available in the art
for generating combinatorial libraries of small organic molecules.
See generally Blondelle et al. Trends Anal. Chem. 14: 83, 1995;
U.S. Pat. Nos. 5,359,115, 5,362,899, 5,288,514, and 5,721,099; Chen
et al. JACS116: 2661, 1994; Kerr et al. JACS115: 252, 1993;
WO92/10092, WO93/09668, WO 94/08051, WO93/20242 and WO91/07087. A
variety of libraries on the order of about 16 to 1,000,000 or more
diversomers can be synthesized and screened for a particular
activity or property using the methods described therein.
[0164] Preferably, the inhibitors finding use in the invention are
small molecules, meaning having a molecular weight of less than
about 1000 Daltons. Such inhibitors are well known in the art. See,
e.g., WO04/07468 and WO04/50022, and U.S. Pat. Nos. 6,710,040;
6,699,871; 6,432,969; 6,303,661; 6,166,063; 6,124,305; 6,110,949;
and 6,107,317, each of which is hereby incorporated by reference in
its entirety. Preferred small molecule inhibitors are orally
effective. DPP-inhibitory antibody or antibody fragments may also
find use in the methods described herein. In this case, antibodies
may be generated to DPP and screened (e.g., using the phage display
methods described herein) to identify antibodies that inhibit DPP
activity on one or more natriuretic peptides of interest.
[0165] Compounds may be screened for inhibitory activity using
isolated DPP enzymes, cell extracts, or blood derived samples as a
source of enzyme, and isolated natriuretic peptides as substrates.
Selection of the conditions to inhibit loss of the penultimate
proline residue from a target natriuretic peptide may depend on the
type of aqueous medium under consideration (for example, inhibition
in a blood sample may require conditions that differ from
inhibition in the circulation of an organism). Selecting such
conditions are within the skill of the artisan. The ability of test
compounds and their corresponding pharmaceutically acceptable acid
addition salts to inhibit DPP may also be demonstrated by employing
a modified version of the assay described in Kubota et al., Clin.
Exp. Immunol. 89: 192-7, 1992. Confirmation of the presence or
absence of the penultimate proline residue may be performed using
an immunoassay selected to be sensitive to the loss of this
N-terminal portion of the molecule, or through the use of mass
spectrometry.
[0166] Proceeding to the next step, candidate compounds that
modulate DPP activity in cultured cells can be tested in animal
models that are relevant to the disease condition of interest. In
these methods, labeled natriuretic peptide may be injected into a
test animal, and the T.sub.1/2 for clearance of the natriuretic
peptide from the circulation may be determined in the presence and
absence of the inhibitor. Preferred animal models of DPP-dependent
natriuretic peptide degradation include rats, mice, sheep, dogs,
cats, and pigs.
[0167] As discussed above, combination treatment with DPP
inhibitors and NEP inhibitors and/or natriuretic peptide(s) is
contemplated by the invention. In addition or as an alternative, a
natriuretic peptide may be provided as an analogue that has been
stabilized to DPP activity, as described for glucose-dependent
insulinotropic polypeptide in Gault et al., Metabolism 52: 679-87,
2003. In preferred embodiments, libraries of natriuretic peptide
analogs having one or more substituted, deleted, added, or modified
amino acids may be screened for improved stability to DPP
degradation. Such analogs preferably retain 50% or more of the
natriuretic activity of the parent natriuretic peptide.
[0168] When administered, the pharmaceutical preparations of the
invention are applied in pharmaceutically-acceptable amounts and in
pharmaceutically-acceptably compositions. Such preparations may
routinely contain salt, buffering agents, preservatives, compatible
carriers, and optionally other therapeutic agents. When used in
medicine, the salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically-acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically
and pharmaceutically-acceptable salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,
salicylic, citric, formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts. The pharmaceutical compositions also may contain,
optionally, suitable preservatives, such as: benzalkonium chloride;
chlorobutanol; parabens and thimerosal. Carrier formulation
suitable for oral, subcutaneous, intravenous, intramuscular, etc.
administrations can be found in Remington's Pharmaceutical
Sciences, Mack Publishing Co., Easton, Pa.
[0169] A variety of administration routes are available for
treating a subject. The particular mode of delivery selected will
depend upon the particular compound selected, the severity of the
condition being treated and the dosage required for therapeutic
efficacy. The methods of the invention, generally speaking, may be
practiced using any mode of administration that is medically
acceptable, meaning any mode that produces effective levels of the
active compounds without causing clinically unacceptable adverse
effects. Such modes of administration include oral, rectal,
topical, nasal, interdermal, intravenous or parenteral routes. Such
modes of administration also include obtaining T cells or bone
marrow cells, stem cells or early lineage progenitor cells from a
patient and contacting the isolated cells with the compounds of the
invention ex vivo, followed by reintroducing the treated cells to
the patient. The treated cells can be reintroduced to the patient
in any manner known in the art for administering viable cells.
[0170] Oral administration is particularly preferred. Compositions
suitable for oral administration may be presented as discrete
units, such as capsules, tablets, lozenges, each containing a
predetermined amount of the compound of the invention. Other
compositions include suspensions in aqueous liquids or non-aqueous
liquids such as a syrup, elixir or an emulsion. Preferably, the
oral preparation does not include an enteric coating since it is
desirable to expose the cyclic compounds of the invention to the
acidic pH conditions of the digestive tract to convert the cyclic
molecules to their linear counterparts.
[0171] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the compounds described above,
increasing convenience to the subject and the physician. Many types
of release delivery systems are available and known to those of
ordinary skill in the art. They include polymer base systems such
as poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono-di- and tri-glycerides; hydrogel
release systems; sylastic systems; peptide based systems; wax
coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which the compound is contained in a form within a matrix such as
those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034
and 5,239,660 and (b) diffusional systems in which an active
component permeates at a controlled rate from a polymer such as
described in U.S. Pat. Nos. 3,832,253, and 3,854,480. In addition,
pump-based hardware delivery systems can be used, some of which are
adapted for implantation.
[0172] Use of a long-term sustained release implant may be
particularly suitable for treatment of chronic conditions.
Long-term release, as used herein, means that the implant is
constructed and arranged to delivery therapeutic levels of the
active ingredient for at least 10 days, and preferably 60 days.
Long-term sustained release implants are well-known to those of
ordinary skill in the art and include some of the release systems
described above.
[0173] The selected compounds are administered in effective
amounts. An effective amount is a dosage of the compound sufficient
to provide a medically desirable result. The effective amount will
vary with the particular condition being treated, the age and
physical condition of the subject being treated, the severity of
the condition, the duration of the treatment, the nature of the
concurrent therapy (if any), the specific route of administration
and like factors within the knowledge and expertise of the health
practitioner. Generally, doses of active compounds will be from
about 0.001 mg/kg per day to 1000 mg/kg per day. It is expected
that doses range of 0.001 to 100 mg/kg will be suitable, preferably
orally and in one or several administrations per day. Lower doses
will result from other forms of administration, such as intravenous
administration. In the event that a response in a subject is
insufficient at the initial doses applied, higher doses (or
effectively higher doses by a different, more localized delivery
route) may be employed to the extent that patient tolerance
permits. Multiple doses per day are contemplated to achieve
appropriate systemic levels of compounds.
EXAMPLES
[0174] The following examples serve to illustrate the present
invention. These examples are in no way intended to limit the scope
of the invention.
Example 1
Blood Sampling
[0175] Blood is preferably collected by venous puncture using a 20
gauge multi-sample needle and evacuated tubes, although fingertip
puncture, plantar surface puncture, earlobe puncture, etc., may
suffice for small volumes. For whole blood collection, blood
specimens are collected by trained study personnel in
EDTA-containing blood collection tubes. For serum collection, blood
specimens are collected by trained study personnel in
thrombin-containing blood collection tubes. Blood is allowed to
clot for 5-10 minutes, and serum is separated from insoluble
material by centrifugation. For plasma collection, blood specimens
are collected by trained study personnel in citrate-containing
blood collection tubes and centrifuged for .gtoreq.12 minutes.
Samples may be kept at 4.degree. C. until use, or frozen at
-20.degree. C. or colder for longer term storage. Whole blood is
preferably not frozen.
Example 2
Recombinant Antibody Preparation
[0176] Immunization of Mice with Antigens and Purification of RNA
from Mouse Spleens
[0177] Mice are immunized by the following method based on
experience of the timing of spleen harvest for optimal recovery of
mRNA coding for antibody. Two species of mice are used: Balb/c
(Charles River Laboratories, Wilmington, Mass.) and A/J (Jackson
Laboratories, Bar Harbor, Me.). Each of ten mice are immunized
intraperitoneally with antigen using 50 .mu.g protein in Freund's
complete adjuvant on day 0, and day 28. Tests bleeds of mice are
obtained through puncture of the retro-orbital sinus. If, by
testing the titers, they are deemed high by ELISA using
biotinylated antigen immobilized via streptavidin, the mice are
boosted with 50 .mu.g of protein on day 70, 71 and 72, with
subsequent sacrifice and splenectomy on day 77. If titers of
antibody are not deemed satisfactory, mice are boosted with 50
.mu.g antigen on day 56 and a test bleed taken on day 63. If
satisfactory titers are obtained, the animals are boosted with 50
.mu.g of antigen on day 98, 99, and 100 and the spleens harvested
on day 105. Typically, a test bleed dilution of 1:3200 or more
results in a half maximal ELISA response to be considered
satisfactory.
[0178] The spleens are harvested in a laminar flow hood and
transferred to a petri dish, trimming off and discarding fat and
connective tissue. Working quickly, spleens are macerated with the
plunger from a sterile 5 cc syringe in the presence of 1.0 ml of
solution D (25.0 g guanidine thiocyanate (Boehringer Mannheim,
Indianapolis, Ind.), 29.3 ml sterile water, 1.76 ml 0.75 M sodium
citrate (pH 7.0), 2.64 ml 10% sarkosyl (Fisher Scientific,
Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol (Fisher Scientific,
Pittsburgh, Pa.)). The spleen suspension is pulled through an 18
gauge needle until viscous and all cells are lysed, then
transferred to a microcentrifuge tube. The petri dish is washed
with 100 .mu.l of solution D to recover any remaining spleen, and
this is transferred to the tube. The suspension is then pulled
through a 22 gauge needle an additional 5-10 times. The sample is
divided evenly between two microcentrifuge tubes and the following
added in order, with mixing by inversion after each addition: 100
.mu.l M sodium acetate (pH 4.0), 1.0 ml water-saturated phenol
(Fisher Scientific, Pittsburgh, Pa.), 200 .mu.l chloroform/isoamyl
alcohol 49:1 (Fisher Scientific, Pittsburgh, Pa.). The solution is
vortexed for 10 seconds and incubated on ice for 15 min. Following
centrifugation at 14,000 rpm for 20 min at 2-8.degree. C., the
aqueous phase is transferred to a fresh tube. An equal volume of
water saturated phenol/chloroform/isoamyl alcohol (50:49:1) is
added, and the tube vortexed for ten seconds. After a 15 min
incubation on ice, the sample is centrifuged for 20 min at
2-8.degree. C., and the aqueous phase transferred to a fresh tube
and precipitated with an equal volume of isopropanol at -20.degree.
C. for a minimum of 30 min. Following centrifugation at 14,000 rpm
for 20 min at 4.degree. C., the supernatant is aspirated away, the
tubes briefly spun and all traces of liquid removed. The RNA
pellets are each dissolved in 300 .mu.l of solution D, combined,
and precipitated with an equal volume of isopropanol at -20.degree.
C. for a minimum of 30 min. The sample is centrifuged 14,000 rpm
for 20 min at 4.degree. C., the supernatant aspirated as before,
and the sample rinsed with 100 .mu.l of ice-cold 70% ethanol. The
sample is again centrifuged 14,000 rpm for 20 min at 4.degree. C.,
the 70% ethanol solution aspirated, and the RNA pellet dried in
vacuo. The pellet is resuspended in 100 .mu.l of sterile distilled
water. The concentration is determined by A260 using an absorbance
of 1.0 for a concentration of 40 .mu.g/ml. The RNA is stored at
-80.degree. C.
[0179] Preparation of Complementary DNA (cDNA)
[0180] The total RNA purified as described above is used directly
as template for preparation of cDNA. RNA (50 .mu.g) is diluted to
100 .mu.L with sterile water, and 10 .mu.L-130 ng/mL oligo
dT.sub.12 is added. The sample is heated for 10 min at 70.degree.
C., then cooled on ice. 40 .mu.L 5.times. first strand buffer is
added (Gibco/BRL, Gaithersburg, Md.), 20 .mu.L 0.1 M dithiothreitol
(Gibco/BRL, Gaithersburg, Md.), 10 .mu.L 20 mM deoxynucleoside
triphosphates (dNTP's, Boehringer Mannheim, Indianapolis, Ind.),
and 10 .mu.L water on ice. The sample is then incubated at
37.degree. C. for 2 min. 10 .mu.L reverse transcriptase
(Superscript.TM.. II, Gibco/BRL, Gaithersburg, Md.) is added and
incubation continued at 37.degree. C. for 1 hr. The cDNA products
are used directly for polymerase chain reaction (PCR).
[0181] Amplification of cDNA by PCR
[0182] To amplify substantially all of the H and L chain genes
using PCR, primers are chosen that corresponded to substantially
all published sequences. Because the nucleotide sequences of the
amino terminals of H and L contain considerable diversity, 33
oligonucleotides are synthesized to serve as 5' primers for the H
chains, and 29 oligonucleotides are synthesized to serve as 5'
primers for the kappa L chains, as described in U.S. 20030104477.
The 5' primers are made according to the following criteria. First,
the second and fourth amino acids of the L chain and the second
amino acid of the heavy chain are conserved. Mismatches that change
the amino acid sequence of the antibody are allowed in any other
position. Second, a 20 nucleotide sequence complementary to the M13
uracil template is synthesized on the 5' side of each primer. This
sequence is different between the H and L chain primers,
corresponding to 20 nucleotides on the 3' side of the pelB signal
sequence for L chain primers and the alkaline phosphatase signal
sequence for H chain primers. The constant region nucleotide
sequences require only one 3' primer each to the H chains and the
kappa L chains (FIG. 2). Amplification by PCR was performed
separately for each pair of 5' and 3' primers. A 50 .mu.L reaction
is performed for each primer pair with 50 pmol of 5' primer, 50
pmol of 3' primer, 0.25 .mu.L Taq DNA Polymerase (5 units/.mu.L,
Boehringer Mannheim, Indianapolis, Ind.), 3 .mu.L cDNA (described
in Example 2), 5 .mu.L 2 mM dNTP's, 5 .mu.L 10.times. Taq DNA
polymerase buffer with MgCl2 (Boehringer Mannheim, Indianapolis,
Ind.), and H.sub.2O to 50 .mu.L. Amplification is done using a
GeneAmp.RTM. 9600 thermal cycler (Perkin Elmer, Foster City,
Calif.) with the following program: 94.degree. C. for 1 min; 30
cycles of 94.degree. C. for 20 sec, 55.degree. C. for 30 sec, and
72.degree. C. for 30 sec; 72.degree. C. for 6 min; 4.degree. C.
[0183] The dsDNA products of the PCR process are then subjected to
asymmetric PCR using only 3' primer to generate substantially only
the anti-sense strand of the target genes. A 100 .mu.L reaction is
done for each dsDNA product with 200 pmol of 3' primer, 2 .mu.L of
ds-DNA product, 0.5 .mu.L Taq DNA Polymerase, 10 .mu.L 2 mM dNTP's,
10 .mu.L 10.times. Taq DNA polymerase buffer with MgCl.sub.2
(Boehringer Mannheim, Indianapolis, Ind.), and H.sub.2O to 100
.mu.L. The same PCR program as that described above is used to
amplify the single-stranded (ss)-DNA.
[0184] Purification of ss-DNA by High Performance Liquid
Chromatography and Kinasing ss-DNA
[0185] The H chain ss-PCR products and the L chain ss-PCR products
are ethanol precipitated by adding 2.5 volumes ethanol and 0.2
volumes 7.5 M ammonium acetate and incubating at -20.degree. C. for
at least 30 min. The DNA is pelleted by centrifuging in an
Eppendorf centrifuge at 14,000 rpm for 10 min at 2-8.degree. C. The
supernatant is carefully aspirated, and the tubes briefly spun a
2nd time. The last drop of supernatant is removed with a pipet. The
DNA is dried in vacuo for 10 min on medium heat. The H chain
products are pooled in 210 .mu.L water and the L chain products are
pooled separately in 210 .mu.L water. The ss-DNA is purified by
high performance liquid chromatography (HPLC) using a Hewlett
Packard 1090 HPLC and a Gen-Pak.TM. FAX anion exchange column
(Millipore Corp., Milford, Mass.) at an oven temperature of
60.degree. C. Absorbance is monitored at 260 nm. The ss-DNA eluted
from the HPLC is collected in 0.5 min fractions. Fractions
containing ss-DNA are ethanol precipitated, pelleted and dried as
described above. The dried DNA pellets are pooled in 200 .mu.L
sterile water.
[0186] If desired, the ss-DNA is kinased on the 5' end in
preparation for mutagenesis. 24 .mu.L 10.times. kinase buffer
(United States Biochemical, Cleveland, Ohio), 10.4 .mu.L 10 mM
adenosine-5'-triphosphate (Boehringer Mannheim, Indianapolis,
Ind.), and 2 .mu.L polynucleotide kinase (30 units/.mu.L, United
States Biochemical, Cleveland, Ohio) is added to each sample, and
the tubes are incubated at 37.degree. C. for 1 hr. The reactions
are stopped by incubating the tubes at 70.degree. C. for 10 min.
The DNA is purified with one extraction of equilibrated phenol
pH>8.0, United States Biochemical, Cleveland,
Ohio)-chloroform-isoamy-1 alcohol (50:49:1) and one extraction with
chloroform:isoamyl alcohol (49:1). After the extractions, the DNA
is ethanol precipitated and pelleted as described above. The DNA
pellets are dried, then dissolved in 50 .mu.L sterile water. The
concentration is determined by measuring the absorbance of an
aliquot of the DNA at 260 nm using 33 .mu.g/mL for an absorbance of
1.0. Samples are stored at -20.degree. C.
[0187] Antibody Phage Display Vector
[0188] The antibody phage display vector for cloning antibodies is
derived from an M13 vector supplied by Ixsys, designated 668-4. The
vector 668-4 contained the DNA sequences encoding the heavy and
light chains of a mouse monoclonal Fab fragment inserted into a
vector described by Huse, WO 92/06024. The vector has a Lac
promoter, a pelB signal sequence fused to the 5' side of the L
chain variable region of the mouse antibody, the entire kappa chain
of the mouse antibody, an alkaline phosphatase signal sequence at
the 5' end of the H chain variable region of the mouse antibody,
the entire variable region and the first constant region of the H
chain, and 5 codons of the hinge region of an IgG 1H chain. A
decapeptide sequence is at the 3' end of the H chain hinge region
and an amber stop codon separates the decapeptide sequence from the
pseudo-gene VIII sequence. The amber stop allows expression of H
chain fusion proteins with the gene VIII protein in E. coli
suppressor strains such as XL1 blue (Stratagene, San Diego,
Calif.), but not in nonsuppressor cell strains such as MK30
(Boehringer Mannheim, Indianapolis, Ind.) (see FIG. 3A).
[0189] To make the first derivative cloning vector, deletions are
made in the variable regions of the H chain and the L chain by
oligonucleotide directed mutagenesis of a uracil template (Kunkel,
Proc. Natl. Acad. Sci. USA 82: 488 (1985); Kunkel, et al., Methods.
Enzymol. 154: 367 (1987)). These mutations delete the region of
each chain from the 5' end of CDR1 to the 3' end of CDR3, and the
mutations add a DNA sequence where protein translation would stop
(see FIG. 4 for mutagenesis oligonucleotides). This prevents the
expression of H or L chain constant regions in clones without an
insert, thereby allowing plaques to be screened for the presence of
insert. The resulting cloning vector is called BS11.
[0190] Many changes are made to BS11 to generate the cloning vector
used in the present screening methods. The amber stop codon between
the heavy chain and the pseudo gene VIII sequence is removed so
that every heavy chain is expressed as a fusion protein with the
gene VIII protein. This increases the copy number of the antibodies
on the phage relative to BS11. A HindIII restriction enzyme site in
the sequence between the 3' end of the L chain and the 5' end of
the alkaline phosphatase signal sequence is deleted so antibodies
can be subcloned into a pBR322 derivative. The interchain cysteine
residues at the carboxyl-terminus of the L and H chains are changed
to serine residues. This increases the level of expression of the
antibodies and the copy number of the antibodies on the phage
without affecting antibody stability. Nonessential DNA sequences on
the 5' side of the lac promoter and on the 3' side of the pseudo
gene VIII sequence are deleted to reduce the size of the M13 vector
and the potential for rearrangement. A transcriptional stop DNA
sequence is added to the vector at the L chain cloning site in
addition to the translational stop so that phage with only heavy
chain proteins on their surface, which might bind nonspecifically
in panning, are not made. Finally, DNA sequences for protein tags
are added to different vectors to allow enrichment for polyvalent
phage by metal chelate chromatography (polyhistidine sequence) or
by affinity purification using a decapeptide tag and a magnetic
latex having an immobilized antibody that binds the decapeptide
tag. The vector BS39 has a polyhistidine sequence at the 3' end of
the kappa chain with no tag at the end of the heavy chain, while
BS45 has a polyhistidine sequence between the end of the heavy
chain constant region and the pseudo-gene VIII sequence, and a
decapeptide sequence at the 3' end of the kappa chain constant
region.
[0191] Preparation of Uracil Templates Used in Generation of Spleen
Antibody Phage Libraries
[0192] 1 mL of E. coli CJ236 (BioRAD, Hercules, Calif.) overnight
culture is added to 50 ml 2.times.YT in a 250 mL baffled shake
flask. The culture is grown at 37.degree. C. to OD.sub.600=0.6,
inoculated with 10 .mu.l of a 1/100 dilution of vector phage stock
and growth continued for 6 hr. Approximately 40 mL of the culture
is centrifuged at 12,000 rpm for 15 minutes at 4.degree. C. The
supernatant (30 mL) is transferred to a fresh centrifuge tube and
incubated at room temperature for 15 minutes after the addition of
15 .mu.l of 10 mg/ml RnaseA (Boehringer Mannheim, Indianapolis,
Ind.). The phage are precipitated by the addition of 7.5 ml of 20%
polyethylene glycol 8000 (Fisher Scientific, Pittsburgh, Pa.)/3.5M
ammonium acetate (Sigma Chemical Co., St. Louis, Mo.) and incubated
on ice for 30 min. The sample is centrifuged at 12,000 rpm for 15
min at 2-8.degree. C. The supernatant is carefully discarded, and
the tube is briefly spun to remove all traces of supernatant. The
pellet is resuspended in 400 .mu.l of high salt buffer (300 mM
NaCl, 100 mM Tris pH 8.0, 1 mM EDTA), and transferred to a 1.5 mL
tube. The phage stock is extracted repeatedly with an equal volume
of equilibrated phenol:chloroform:isoamyl alcohol (50:49:1) until
no trace of a white interface is visible, and then extracted with
an equal volume of chloroform:isoamyl alcohol (49:1). The DNA is
precipitated with 2.5 volumes of ethanol and 1/5 volume 7.5 M
ammonium acetate and incubated 30 min at -20.degree. C. The DNA is
centrifuged at 14,000 rpm for 10 min at 4.degree. C., the pellet
washed once with cold 70% ethanol, and dried in vacuo. The uracil
template DNA is dissolved in 30 .mu.l sterile water and the
concentration determined by A260 using an absorbance of 1.0 for a
concentration of 40 .mu.g/ml. The template is diluted to 250
ng/.mu.l with sterile water, aliquoted, and stored at -20.degree.
C.
[0193] Mutagenesis of Uracil Template with Ss-DNA and
Electroporation into E. Coli to Generate Antibody Phage
Libraries
[0194] Antibody phage-display libraries are generated by
simultaneously introducing single-stranded heavy and light chain
genes onto a phage-display vector uracil template. A typical
mutagenesis is performed on a 2 .mu.g scale by mixing the following
in a 0.2 mL PCR reaction tube: 8 .mu.l of (250 ng/.mu.l) uracil
template (examples 5 and 6), 8 .mu.l of 10.times. annealing buffer
(200 mM Tris pH 7.0, 20 mM MgCl.sub.2, 500 mM NaCl), 3.33 .mu.l of
kinased single-stranded heavy chain insert (100 ng/.mu.l), 3.1
.mu.l of kinased single-stranded light chain insert (100 ng/ml),
and sterile water to 80 .mu.l. DNA is annealed in a GeneAmp.RTM.
9600 thermal cycler using the following thermal profile: 20 sec at
94.degree. C., 85.degree. C. for 60 sec, 85.degree. C. to
55.degree. C. ramp over 30 min, hold at 55.degree. C. for 15 min.
The DNA is transferred to ice after the program finishes. The
extension/ligation is carried out by adding 8 .mu.l of 10.times.
synthesis buffer (5 mM each dNTP, 10 mM ATP, 100 mM Tris pH 7.4, 50
mM MgCl.sub.2, 20 mM DTT), 8 .mu.l T4 DNA ligase (1 U/.mu.l,
Boehringer Mannheim, Indianapolis, Ind.), 8 .mu.l diluted T7 DNA
polymerase (1 U/.mu.l, New England BioLabs, Beverly, Mass.) and
incubated at 37.degree. C. for 30 min. The reaction is stopped with
300 .mu.l of mutagenesis stop buffer (10 mM Tris pH 8.0, 10 mM
EDTA). The mutagenesis DNA is extracted once with equilibrated
phenol (pH>8):chloroform:isoamyl alcohol (50:49:1), once with
chloroform:isoamyl:alcohol (49:1), and the DNA is ethanol
precipitated at -20.degree. C. for at least 30 min. The DNA is
pelleted and the supernatant carefully removed as described above.
The sample is briefly spun again and all traces of ethanol removed
with a pipetman. The pellet is dried in vacuo. The DNA is
resuspended in 4 .mu.l of sterile water.
[0195] 1 .mu.l mutagenesis DNA is (500 ng) is transferred into 40
.mu.l electrocompetent E. coli DH12S (Gibco/BRL, Gaithersburg,
Md.). The transformed cells are mixed with 1.0 mL 2.times.YT broth
and transferred to 15 mL sterile culture tubes. The first round
antibody phage is made by shaking the cultures overnight at
23.degree. C. and 300 rpm. The efficiency of the electroporation is
measured by plating 10 .mu.l of 10.sup.-3 and 10.sup.-4 dilutions
of the cultures on LB agar plates. These plates are incubated
overnight at 37.degree. C. The efficiency is determined by
multiplying the number of plaques on the 10.sup.-3 dilution plate
by 10.sup.5 or multiplying the number of plaques on the 10.sup.-4
dilution plate by 10.sup.6. The overnight cultures from the
electroporations are transferred to 1.5 ml tubes, and the cells are
pelleted by centrifuging at 14,000 rpm for 5 min. The supernatant,
which is the first round of antibody phage, is then transferred to
15 mL sterile centrifuge tubes with plug seal caps.
[0196] Transformation of E. coli by Electroporation
[0197] The electrocompetent E. coli cells are thawed on ice. DNA is
mixed with 20-40 .mu.L electrocompetent cells by gently pipetting
the cells up and down 2-3 times, being careful not to introduce
air-bubbles. The cells are transferred to a Gene Pulser cuvette
(0.2 cm gap, BioRAD, Hercules, Calif.) that has been cooled on ice,
again being careful not to introduce an air-bubble in the transfer.
The cuvette is placed in the E. coli Pulser (BioRAD, Hercules,
Calif.) and electroporated with the voltage set at 1.88 kV
according to the manufacturer's recommendation. The transformed
sample is immediately diluted to 1 ml with 2.times.YT broth and
processed as procedures dictate.
[0198] Preparation of Biotinylated Antigens and Antibodies
[0199] Protein antigens or antibodies are dialyzed against a
minimum of 100 volumes of 20 mM borate, 150 mM NaCl, pH 8 (BBS) at
2-8.degree. C. for at least 4 hr. The buffer is changed at least
once prior to biotinylation. Protein antigens or antibodies are
reacted with biotin-XX-NHS ester (Molecular Probes, Eugene, Oreg.,
stock solution at 40 mM in dimethylformamide) at a final
concentration of 1 mM for 1 hr at room temperature. After 1 hr, the
protein antigens or antibodies are extensively dialyzed into BBS to
remove unreacted small molecules.
[0200] Preparation of Alkaline Phosphatase-Antigen Conjugates
[0201] Alkaline phosphatase (AP, Calzyme Laboratories, San Luis
Obispo, Calif.) is placed into dialysis versus a minimum of 100
volumes of column buffer (50 mM potassium phosphate, 10 mM borate,
150 mM NaCl, 1 mM MgSO.sub.4, pH 7.0) at 2-8.degree. C. for at
least four hr. The buffer is changed at least twice prior to use of
the AP. When the AP is removed from dialysis and brought to room
temperature, the concentration is determined by absorbance at 280
nm using an absorbance of 0.77 for a 1 mg/mL solution. The AP is
diluted to 5 mg/mL with column buffer. The reaction of AP and
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC,
Pierce Chemical Co., Rockford, Ill.) is carried out using a 20:1
ratio of SMCC:AP. SMCC is dissolved in acetonitrile at 20 mg/mL and
diluted by a factor of 84 when added to AP while vortexing or
rapidly stirring. The solution is allowed to stand at room
temperature for 90 min before the unreacted SMCC and low molecular
weight reaction products are separated from the AP using gel
filtration chromatography (G50 Fine, Pharmacia Biotech, Piscataway,
N.J.) in a column equilibrated with column buffer.
[0202] Protein antigen is dialyzed versus a minimum of 100 volumes
of 20 mM potassium phosphate, 4 mM borate, 150 mM NaCl, pH 7.0 at
2-8.degree. C. for at least four hr. The buffer is changed at least
twice prior to use of the antigen. The amount of antigen is
quantitated by absorbance at 280 nm or by the method of Lowry. The
reaction of antigen and N-succinimidyl
3-[2-pyridyldithio]propionate (SPDP, Pierce Chemical Co., Rockford,
Ill.) is carried out using a 20:1 molar ratio of SPDP:antigen. SPDP
is dissolved in dimethylformamide at 40 mM and diluted into the
antigen solution while vortexing. The solution is allowed to stand
at room temperature for 90 min, at which time the reaction is
quenched by adding taurine (Aldrich Chemical Co., Milwaukee, Wis.)
to a final concentration of 20 mM for 5 min. Dithiothreitol (Fisher
Scientific, Pittsburgh, Pa.) is added to the protein at a final
concentration of 1 mM for 30 min. The low molecular weight reaction
products are separated from the antigen using gel filtration
chromatography in a column equilibrated in 50 mM potassium
phosphate, 10 mM borate, 150 mM NaCl, 0.1 mM ethylene diamine
tetraacetic acid (EDTA, Fisher Scientific, Pittsburgh, Pa.), pH
7.0.
[0203] The AP and antigen are mixed together in an equimolar ratio.
The reaction is allowed to proceed at room temperature for 2 hr.
The conjugate is diluted to 0.1 mg/mL with block containing 1%
bovine serum albumin (from 30% BSA, Bayer, Kankakee, Ill.), 10 mM
Tris, 150 mM NaCl, 1 mM MgCl.sub.2, 0.1 mM ZnCl.sub.2, 0.1%
polyvinyl alcohol (80% hydrolyzed, Aldrich Chemical Co., Milwaukee,
Wis.), pH 8.0.
[0204] Preparation of Avidin Magnetic Latex
[0205] Magnetic latex (Estapor, 10% solids, Bangs Laboratories,
Fishers, Ind.) is thoroughly resuspended and 2 ml aliquoted into a
15 ml conical tube. The magnetic latex is suspended in 12 ml
distilled water and separated from the solution for 10 min using a
magnet. While still in the magnet, the liquid is carefully removed
with a 10 mL sterile pipet. This washing process is repeated an
additional three times. After the final wash, the latex is
resuspended in 2 ml of distilled water. In a separate 50 ml conical
tube, 10 mg of avidin-HS (NeutrAvidin, Pierce, Rockford, Ill.) is
dissolved in 18 ml of 40 mM Tris, 0.15 M sodium chloride, pH 7.5
(TBS). While vortexing, the 2 ml of washed magnetic latex is added
to the diluted avidin-HS and the mixture vortexed an additional 30
seconds. This mixture is incubated at 45.degree. C. for 2 hr,
shaking every 30 minutes. The avidin magnetic latex is separated
from the solution using a magnet and washed three times with 20 ml
BBS as described above. After the final wash, the latex is
resuspended in 10 ml BBS and stored at 4.degree. C.
[0206] Immediately prior to use, the avidin magnetic latex is
equilibrated in panning buffer (40 mM TRIS, 150 mM NaCl, 20 mg/mL
BSA, 0.1% Tween 20 (Fisher Scientific, Pittsburgh, Pa.), pH 7.5).
The avidin magnetic latex needed for a panning experiment (200
.mu.l/sample) is added to a sterile 15 ml centrifuge tube and
brought to 10 ml with panning buffer. The tube is placed on the
magnet for 10 min to separate the latex. The solution is carefully
removed with a 10 mL sterile pipet as described above. The magnetic
latex is resuspended in 10 mL of panning buffer to begin the second
wash. The magnetic latex is washed a total of 3 times with panning
buffer. After the final wash, the latex is resuspended in panning
buffer to the initial aliquot volume.
[0207] Plating M13 Phage or Cells Transformed with Antibody
Phage-Display Vector Mutagenesis Reaction
[0208] The phage samples are added to 200 .mu.L of an overnight
culture of E. coli XL 1-Blue when plating on 100 mm LB agar plates
or to 600 .mu.L of overnight cells when plating on 150 mm plates in
sterile 15 ml culture tubes. After adding LB top agar (3 mL for 100
mm plates or 9 mL for 150 mm plates, top agar stored at 55.degree.
C., Appendix A1, Molecular Cloning, A Laboratory Manual, (1989)
Sambrook. J), the mixture is evenly distributed on an LB agar plate
that had been pre-warmed (37.degree. C.-55.degree. C.) to remove
any excess moisture on the agar surface. The plates are cooled at
room temperature until the top agar solidified. The plates are
inverted and incubated at 37.degree. C. as indicated.
[0209] Develop Nitrocellulose Filters with Alkaline Phosphatase
Conjugates
[0210] After overnight incubation of the nitrocellulose filters on
LB agar plates, the filters are carefully removed from the plates
with membrane forceps and incubated for 2 hr in either casein block
(block with 1% casein (Hammersten grade, Research Organics,
Cleveland, Ohio)), when using antigen-AP conjugates or block when
using goat anti-mouse kappa-AP (Southern Biotechnology Associates,
Inc, Birmingham, Ala.). After 2 hr, the filters are incubated with
the AP conjugate for 2-4 hr. Antigen-AP conjugates are diluted into
casein block at a final concentration of 1 .mu.g/mL and goat
anti-mouse kappa-AP conjugates are diluted into block at a final
concentration of 1 .mu.g/mL. Filters are washed 3 times with 40 mM
TRIS, 150 mM NaCl, 0.05% Tween 20, pH 7.5 (TBST) (Fisher Chemical,
Pittsburgh, Pa.) for 5 min each. After the final wash, the filters
are developed in a solution containing 0.2 M
2-amino-2-methyl-1-propanol (JBL Scientific, San Luis Obispo,
Calif.), 0.5 M TRIS, 0.33 mg/mL nitro blue tetrazolium (Fisher
Scientific, Pittsburgh, Pa.) and 0.166 mg/mL
5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt.
[0211] Enrichment of Polyclonal Phage to BNP Peptides with No Tags
on the Heavy Chain and a Polyhistidine Sequence on the Kappa
Chain
[0212] This example describes multiple rounds of screening of a
phage library to BNP peptides. Some of the rounds of screening are
alternated with rounds of enrichment for phage displaying multiple
copies of antibodies. The percentage of phage displaying any light
chain, and the percentage of phage displaying Fab fragments with
specific affinity for BNP peptides of interest (referred to below
as "antigen") is measured after each round of screening.
[0213] The first round antibody phage is prepared as described
above using BS39 uracil template. Two electroporations of
mutagenesis DNA had efficiencies of 9.7.times.10.sup.7 PFU and
8.3.times.10.sup.7 PFU. The phage from both electroporations are
combined and diluted to 3.2 ml with panning buffer. The phage is
aliquoted into 2-1 mL aliquots in 15 mL disposable sterile
centrifuge tubes with plug seal caps. Antigen-biotin (10 .mu.L,
10.sup.-6 M stock concentration) is added to each phage aliquot.
The phage samples are incubated overnight at 2-8.degree. C.
[0214] After the incubation, the phage samples are panned with
avidin magnetic latex. The equilibrated avidin magnetic latex (see
Example 11), 200 .mu.L latex per sample, is incubated with the
phage for 10 min at room temperature. After 10 min, approximately 9
mL of panning buffer is added to each phage sample, and the
magnetic latex is separated from the solution using a magnet. After
10 min in the magnet, the unbound phage is carefully removed with a
10 mL sterile pipet. The magnetic latex is then resuspended in 10
mL of panning buffer to begin the second wash. The latex is washed
a total of 5 times as described above. For each wash, the tubes are
in the magnet for 10 min to separate unbound phage from the
magnetic latex. After the 5th wash, the magnetic latex is
resuspended in 1 mL TBS and transferred to a 1.5 mL tube. Aliquots
of the latex are taken at this point to plate on 100 mm LB agar
plates as described above. The bulk of the magnetic latex (99%) is
resuspended in 200 .mu.L 2.times.YT and is plated on a 150 mm LB
agar plate as described in Example 12. The 100 mm LB agar plates
are incubated at 37.degree. C. for 6-7 hr, then the plates are
transferred to room temperature and nitrocellulose filters (pore
size 0.45 .mu.m, BA85 Protran, Schleicher and Schuell, Keene, N.H.)
are overlayed onto the plaques. Plates with nitrocellulose filters
are incubated overnight at room temperature. The 150 mm plates are
used to amplify the phage binding to the magnetic latex to generate
the next round of antibody phage. These plates are incubated at
37.degree. C. for 4 hr, then overnight at 20.degree. C.
[0215] After the overnight incubation, the antibody phage is eluted
from the 150 mm plates, and the filters are developed with alkaline
phosphatase-antigen as described herein. The antibody phage is
eluted from the 150 mm plates by pipeting 8 mL 2YT media onto the
lawn and gently shaking the plate at room temperature for 20 min.
The phage are transferred to a 15 mL disposable sterile centrifuge
tubes with plug seal cap and the debris from the LB plate is
pelleted by centrifuging for 15 min at 3500 rpm. The 2nd round
antibody phage is then transferred to a new tube.
[0216] To begin the 2nd round of panning, the antibody phage are
titered by plating 10 .mu.L of 10.sup.-7 and 10.sup.-8 dilutions of
the phage on 100 mm LB agar plates. The plates are incubated at
37.degree. C. for 6-7 hr, then the number of plaques on the plates
are counted. Also, to monitor the percentage of kappa positives in
the antibody phage, a nitrocellulose filter is overlayed onto the
plate and incubated overnight at room temperature. The percentage
of kappa positives is a measure of the proportion of phage
displaying intact Fab fragments.
[0217] Both 2nd round antibody phage samples are pooled by diluting
each sample into panning buffer at a final concentration of
5.times.10.sup.9 PFU/mL to a final volume of 1 mL. (The titers of
the antibody phage are about 2.times.10.sup.12 PFU/mL and
1.7.times.10.sup.12). Antigen-biotin (10 .mu.L, 10.sup.-6 M stock
concentration) is added to the phage and the phage is incubated at
2-8.degree. C. overnight. The nitrocellulose filters on the
antibody phage titer plates are developed with goat anti-mouse
kappa AP as described herein. The second round antibody phage is
panned with avidin magnetic latex as described above. After washing
the latex with panning buffer, the latex is resuspended in 1 mL TBS
and transferred to a 1.5 mL tube. Aliquots of the latex are plated
on 100 mm LB agar plates as described above to check functional
positives, and the rest of the latex is plated on 150 mm LB agar
plates to generate the 3rd round antibody phage. This general
procedure of tittering the antibody phage, diluting the phage into
panning buffer and adding antigen-biotin, incubating the phage at
least 16 hr at 2-8.degree. C., panning the phage with avidin
magnetic latex, and plating the magnetic latex is followed through
10 rounds of panning. The only changes from that described above is
the concentration of antigen-biotin is lower to increase the
affinity of bound antibodies, and the number of phage panned is
between 10.sup.10 and 10.sup.8.
[0218] After the 10th round of panning to antigen-biotin, the
antibody phage are subject to a round of enrichment for polyvalent
display. Enrichment is effected by binding of the hexahistidine tag
fused to the displayed light chain to Ni NTA agarose (Qiagen Inc.,
Chatsworth, Calif.). The 11 th round antibody phage (2.5 mL) are
diluted into 2.5 mL panning buffer in a 15 mL disposable sterile
centrifuge tube with plug seal cap. The Ni NTA is equilibrated into
panning buffer using the following procedure. The resin (1 mL per
phage sample) is diluted to 50 mL with panning buffer in a 50 mL
disposable sterile centrifuge tube with plug seal cap and then is
pelleted in an IEC centrifuge at 500 rpm for 1 min. The supernatant
is carefully removed with a 50 mL disposable pipet, and the resin
is again diluted to 50 mL with panning buffer for the second wash.
The resin is washed in this manner a total of 4 times in order to
equilibrate the resin in panning buffer. The equilibrated resin is
then resuspended to its original volume with panning buffer.
Equilibrated resin (1 mL) is then added to the phage, and the tube
is gently rocked for 15 min. After 15 min, the resin is pelleted in
an IEC centrifuge at 500 rpm for 1 min. The supernatant is gently
removed with a 10 mL disposable pipet, and the resin is resuspended
in 10 mL panning buffer for the first wash. The resin is pelleted
as described above, the supernatant is removed, and the resin is
resuspended a 2nd time in 10 mL panning buffer. This procedure is
repeated for a total of 5 panning buffer washes. After the 5th wash
is removed, the resin is resuspended in 1 mL of elution buffer (50
mM citrate, 150 mM NaCl, pH 4.0) and transferred to a 1.5 mL tube.
The resin is gently rocked for 1 hr to elute the antibody phage.
After 1 hr, the resin is pelleted (14,000 rpm in Eppendorf
centrifuge for 5 min), and the phage is removed while being careful
not to transfer any resin. In order to adjust the pH of the phage
solution to 8, 50 .mu.L of 1 M Tris, pH 8.3 and 46 .mu.L of 1 M
NaOH are added to the 1 mL phage sample. Also, 10 1 L of 300 mg/mL
bovine serum albumin (Bayer, Kankakee, Ill.) is added to the phage
sample. The resulting phage solution (1 mL) is transferred to a 15
mL disposable sterile centrifuge tube with plug seal cap for the
11th round of panning with antigen-biotin, as described above.
[0219] The 12th-14th rounds of panning are done as described above,
where the antibody phage is bound to Ni NTA, eluted, and the eluted
phage panned with antigen-biotin. However, in round 13, unlabelled
C-terminal BNP peptides are added to the phage eluted from the Ni
NTA at 100-fold molar excess to the antigen-biotin to select
antibodies that specifically bind to antigen without binding to the
C-terminal BNP peptides.
Example 3
Biochemical Analyses
[0220] BNP is measured using standard immunoassay techniques. These
techniques involve the use of antibodies to specifically bind the
protein targets. An antibody directed against BNP is biotinylated
using N-hydroxysuccinimide biotin (NHS-biotin) at a ratio of about
5 NHS-biotin moieties per antibody. The biotinylated antibody is
then added to wells of a standard avidin 384 well microtiter plate,
and biotinylated antibody not bound to the plate is removed. This
formed an anti-BNP solid phase in the microtiter plate. Another
anti-BNP antibody is conjugated to alkaline phosphatase using
standard techniques, using SMCC and SPDP (Pierce, Rockford, Ill.).
The immunoassays are performed on a TECAN Genesis RSP 200/8
Workstation. Test samples (10 .mu.L) are pipeted into the
microtiter plate wells, and incubated for 60 min. The sample is
then removed and the wells washed with a wash buffer, consisting of
20 mM borate (pH 7.42) containing 150 mM NaCl, 0.1% sodium azide,
and 0.02% Tween-20. The alkaline phosphatase-antibody conjugate is
then added to the wells and incubated for an additional 60 min,
after which time, the antibody conjugate is removed and the wells
washed with a wash buffer. A substrate, (AttoPhos.RTM., Promega,
Madison, Wis.) is added to the wells, and the rate of formation of
the fluorescent product is related to the concentration of the BNP
in the test samples.
Example 4
Synthesis of DPP Inhibitors
[0221] Peptide coupling chemistry is preferably employed to prepare
linear boroPro compounds. The peptide coupling chemistry methods
and procedures used in this invention are readily available.
Examples of books using these methods include, but are not limited
to, the following citations incorporated herein by reference: P. D.
Bailey, "An Introduction to Peptide Chemistry," John Wiley &
Sons, 1990; Miklos Bodansky, "Peptide Chemistry A Practical
Textbook," Springer-Verlag, 1988; Miklos Bodansky, "Principles of
Peptide Synthesis, Reactivity and Structure Concepts in Organic
Chemistry," Volume 16, Springer-Verlag, 1984; and Miklos Bodansky,
"Principles of Peptide Synthesis, Reactivity and Structure Concepts
in Organic Chemistry," Volume 21, Springer-Verlag, 1984.
[0222] The compounds of the invention can begin with the synthesis
of H-boroPro as disclosed in WO 98/00439. Use of H-boroPro is for
illustrative purposes only, and is not intended to limit the scope
of this invention. According to WO 98/00439, H-boroPro may be
prepared by the synthetic route previously developed and described
(G. R. Flentke, et al., "Inhibition of dipeptidyl aminopeptidase IV
(DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to
examine the role of DP-IV in T-cell function," PNAS (U.S.A.) 88,
1556-1559 (1991); also described in U.S. Pat. No. 5,462,928).
Alternatively, H-boroPro may be produced by a new procedure (Kelly,
T. A., et al., "The efficient synthesis and simple resolution of a
proline boronate ester suitable for enzyme inhibition studies,"
Tetrahedron 49, 1009-1016 (1993)). Both of these synthetic routes
reportedly yield racemic H-boroPro pinanediol.
[0223] According to WO 98/00439, stereochemically pure L, L and L,
D diastereomers of Z-Lys-boroPro are prepared by first resolving
racemic H-boroPro through crystallization with optically active
blocking protecting groups ((1S, 2S, 3R, 5S)-+-pinanediol isomer)
followed by coupling the isotopically pure L-boroPro and D-boroPro
to the stereochemically pure L isomer of lysine (See U.S. Pat. No.
5,462,928). Alternatively, the L,L and L,D diastereomers of
Lys-boroPro are prepared in high optical purity by coupling racemic
H-boroPro by L-Lys and separating the resulting diastereomeric
Z-Lys-boroPro-diester into its component L,D and L,L diastereomers
using reverse phase HPLC as previously described for diastereomeric
Pro-boroPro (W. G. Gutheil and W. W. Bachovchin, "Separation of
L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic
Analysis of Their Inhibition of Dipeptidyl Peptidase IV. A New
Method for the Analysis of Slow, Tight-Binding Inhibition,"
Biochemistry 32, 8723-8731 (1993)).
Example 5
Purification of Dipeptidyl Peptidase
[0224] The following examples are exemplary for dipeptidyl
peptidase IV; dipeptidyl peptidase II may be isolated and used
similarly according to the methods of U.S. Pat. No. 6,485,955.
[0225] Porcine enzyme is purified as previously described (1), with
several modifications. Kidneys from 15-20 animals are obtained, and
the cortex dissected away and frozen at -80.degree. C. Frozen
tissue (2000-2500 g) is homogenized in 12 L of 0.25 M sucrose in a
Waring blender. The homogenate is left at 37.degree. C. for 18
hours to facilitate cleavage of DPP4 from cell membranes. After the
cleavage step, the homogenate is clarified by centrifugation at
7000.times.g for 20 minutes at 4.degree. C., and the supernatant is
collected. Solid ammonium sulfate is added to 60% saturation, and
the precipitate is collected by centrifugation at 10,000.times.g
and discarded. Additional ammonium sulfate is added to the
supernatant to 80% saturation, and the 80% pellet is collected and
dissolved in 20 mM Na.sub.2HPO.sub.4, pH 7.4.
[0226] After dialysis against 20 mM Na.sub.2HPO.sub.4, pH 7.4, the
preparation is clarified by centrifugation at 10,000.times.g. The
clarified preparation then is applied to 300 ml of ConA Sepharose
equilibrated in the same buffer. After washing with buffer to a
constant A280, the column is eluted with 5% (wt/vol) methyl
.alpha.-D-mannopyranoside. Active fractions are pooled,
concentrated, and dialyzed against 5 mM sodium acetate, pH 5.0.
Dialyzed material is flowed through a 100 ml Pharmacia Resource S
column equilibrated in the same buffer. The flowthrough material is
collected and contained most of the enzyme activity. Active
material again is concentrated and dialyzed into 20 mM
Na.sub.2HPO.sub.4, pH 7.4. Lastly, the concentrated enzyme is
chromatographed on a Pharmacia S-200 gel filtration column to
removed low molecular weight contaminants. Purity of column
fractions is analyzed by reducing SDS-PAGE, and the purest
fractions pooled and concentrated. Purified enzyme is stored in 20%
glycerol at -80.degree. C.
Example 6
Assay of Dipeptidyl Peptidase
[0227] Enzyme is assayed under steady-state conditions as
previously described in Nagatsu et al., Anal. Biochem. 74: 466-76,
1976 with BNP as substrate, with the following modifications.
Reactions contain, in a final volume of 100 .mu.L, 100 mM ACES, 52
mM TRIS, 52 mM ethanolamine, 500 .mu.M substrate, 0.2% DMSO, and
4.5 nM enzyme at 25.degree. C., pH 7.4. For analysis of positive
compounds, steady-state kinetic inhibition constants are determined
as a function of both substrate and inhibitor concentration.
Complete inhibition experiments contain 11 substrate and 7
inhibitor concentrations, with triplicate determinations. For tight
binding inhibitors with K.sub.i s less than 20 nM, the enzyme
concentration is reduced to 0.5 nM and reaction times are increased
to 120 minutes. Pooled datasets from the three plates are fitted to
the appropriate equation for either competitive, noncompetitive or
uncompetitive inhibition.
Example 7
Chemical Deglycosylation of Naturietic Peptides
[0228] 50 .mu.g of protein is incubated at 4.degree. C. for 2 hours
in the dark, with 100 .mu.l of trifluoromethanesulfonic
acid/anisole (2:1, v/v) reagent in glass tubing saturated with
N.sub.2. The reaction is stopped by the addition of 60% (v/v)
pyridine at -20.degree. C. The protein is extensively dialysed
against 25 mM sodium phosphate buffer pH 7.0.
Example 8
Enzymatic Deglycosylation of Naturietic Peptides
[0229] To 40 .mu.L of sample is added 2 .mu.L 25.times. protease
inhibitor cocktail (Sigma cat. # P2714), 1 .mu.L O-glycanase
(Prozyme cat. # GK80090), 3 .mu.L sialidase A (Prozyme cat. #
GK80040), and 8 .mu.L 200 mM sodium phosphate buffer pH 7,0. The
mixture is vortexed gently and incubated at 20.degree. C. for 4
hours.
Example 9
Analysis of Natriuretic Peptides by Mass Spectrometry
[0230] Preparation of Antibody Capture Surface
[0231] 3 .mu.L of antibody solution (0.25 mg/mL anti-BNP monoclonal
antibody in borate buffered saline pH 8.0 ("BBS")) is applied to
appropriate spots of a PS10 ProteinChip array (Ciphergen cat. #
C553-0044), and the chip is placed in a humid chamber with gentle
agitation at 20.degree. C. for 3 hours. The antibody solution is
removed, and the array spots are washed twice with 3 .mu.L of 1.5
mg/mL BSA/0.1% Triton X-100/0.5 M Tris-HCl pH 8.0. At the second
wash, the chip is placed in a humid chamber without agitation at
20.degree. C. for 3 hours. Following this wash, the array is
immersed in 5 mM HEPES pH 7.5, and the excess buffer is
removed.
[0232] Capture of BNP
[0233] Using a BIOMEC robotic pipetting station (Beckman
Instruments), the array is washed with 150 .mu.L 1% Triton X-100 in
BBS for 10 minutes; 150 .mu.L 10% PEG300/0.1% Triton X-100 in BBS
for 10 minutes; and 3.times. with 150 .mu.L 0.2% Triton X-100 in
BBS for 5 minutes each. 40 .mu.L 0.2% Triton X-100 in BBS and 40
.mu.L deglycosylated sample (or control sample) is applied and
incubated overnight at 4.degree. C. with gentle agitation.
[0234] Application of Energy Absorbing Matrix and MS Analysis
[0235] Following this incubation, the array is washed 3.times. with
150 .mu.L 1M urea/0.1% CHAPS/0.3M KCl/50 mM Tris-HCl pH 7.5 for 1
minute each; and 3.times. with 300 .mu.L 5 mM HEPES pH 7.5 for 3
seconds each. Excess buffer is removed, and the array is allowed to
air dry until no sheen is visible. For low molecular weight
analysis (M/Z<6000), 1 .mu.L of 20%
.alpha.-cyano-4-hydroxycinnamic acid (CHCA, Ciphergen cat. #
C300-0001) in 0.5% trifluoroacetic acid (Pierce cat #28904)/50%
acetonitrile (Pierce cat. # 20062) is applied to appropriate spots
as an energy absorbing matrix ("EAM"). For high molecular weight
analysis (M/Z.gtoreq.6000), 1 .mu.L of 50% sinapinic acid (SPA,
Ciphergen Cat. No. C300-0002) in 0.5% trifluoroacetic acid/50%
acetonitrile is applied to appropriate spots as an EAM. Spots are
allowed to air dry, and a second 1 .mu.L drop of the appropriate
EAM is applied.
[0236] MS spectra are acquired using a Ciphergen ProteinChip reader
model PBS IIC. For low molecular weight analysis, the following
instrument parameters are used: high mass is set to 70 kDa
optimized from 2 kDa to 15 kDa; starting laser intensity is set to
165; starting detector sensitivity is set to 9; mass deflector is
set to 1 kDa; acquisition method is set to SELDI quantitation;
SELDI acquisition parameters=26, delta=10, transients per=18,
ending position=76; and warming positions with 5 shots at
intensity=175. For high molecular weight analysis, the following
instrument parameters are used: high mass is set to 70 kDa
optimized from 3 kDa to 30 kDa; starting laser intensity is set to
200; starting detector sensitivity is set to 9; mass deflector is
set to 2 kDa; acquisition method is set to SELDI quantitation;
SELDI acquisition parameters=24, delta=10, transients per=13,
ending position=74; and warming positions with 3 shots at
intensity=210.
Example 10
Inhibition of BNP Degradation
[0237] Two human plasma samples were individually divided into two
tubes, to one of which the reversible DPP inhibitor Diprotin A
(Ile-Pro-Ile) was added to a concentration of 1 mM. Each sample was
spiked with human BNP.sub.77-108, and the mixture held at 4.degree.
C. overnight. Each sample was subjected to antibody capture SELDI
mass spectrometry as described above. The results are depicted in
FIG. 1. Panels A (no addition) and B (Diprotin A) represent one
plasma sample, and panels C (no addition) and D (Diprotin A) the
second plasma sample. Capture was performed with an antibody that
recognizes human BNP.
[0238] Full length BNP.sub.77-108 would be expected to appear at a
molecular weight of about 3466 Da (large arrowhead), and
BNP.sub.79-108 (in which cleavage occurs following the penultimate
proline) at about 3282 Da (small arrowhead). Comparing panels A to
B and C to D, the cleavage of BNP.sub.77-108 to BNP.sub.79-108 is
inhibited by the DPP inhibitor. A second cleavage product, presumed
to be BNP.sub.79-106 at a molecular weight of about 2988 Da, is
also inhibited by the Diprotin A treatment, with a corresponding
increase in a cleavage product presumed to be BNP.sub.77-106 at a
molecular weight of about 3173 Da. Thus, removal of the amino
terminal ser-pro dipeptide is sensitive to the presence of a DPP
inhibitor, while a second carboxyl terminal dipeptide cleavage is
not.
[0239] While the invention has been described and exemplified in
sufficient detail for those skilled in this art to make and use it,
various alternatives, modifications, and improvements should be
apparent without departing from the spirit and scope of the
invention.
[0240] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The examples provided herein are representative of preferred
embodiments, are exemplary, and are not intended as limitations on
the scope of the invention. Modifications therein and other uses
will occur to those skilled in the art. These modifications are
encompassed within the spirit of the invention and are defined by
the scope of the claims.
[0241] It will be readily apparent to a person skilled in the art
that varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0242] All patents and publications mentioned in the specification
are indicative of the levels of those of ordinary skill in the art
to which the invention pertains. All patents and publications are
herein incorporated by reference to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0243] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
[0244] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
51108PRTHomo sapiens 1His Pro Leu Gly Ser Pro Gly Ser Ala Ser Asp
Leu Glu Thr Ser Gly1 5 10 15Leu Gln Glu Gln Arg Asn His Leu Gln Gly
Lys Leu Ser Glu Leu Gln 20 25 30Val Glu Gln Thr Ser Leu Glu Pro Leu
Gln Glu Ser Pro Arg Pro Thr 35 40 45Gly Val Trp Lys Ser Arg Glu Val
Ala Thr Glu Gly Ile Arg Gly His 50 55 60Arg Lys Met Val Leu Tyr Thr
Leu Arg Ala Pro Arg Ser Pro Lys Met65 70 75 80Val Gln Gly Ser Gly
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser 85 90 95Ser Ser Gly Leu
Gly Cys Lys Val Leu Arg Arg His 100 1052134PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Met Asp Pro Gln Thr Ala Pro Ser Arg Ala Leu Leu Leu Leu Leu Phe1 5
10 15Leu His Leu Ala Phe Leu Gly Gly Arg Ser His Pro Leu Gly Ser
Pro 20 25 30Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly Leu Gln Glu Gln
Arg Asn 35 40 45His Leu Gln Gly Lys Leu Ser Glu Leu Gln Val Glu Gln
Thr Ser Leu 50 55 60Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr Gly Val
Trp Lys Ser Arg65 70 75 80Glu Val Ala Thr Glu Gly Ile Arg Gly His
Arg Lys Met Val Leu Tyr 85 90 95Thr Leu Arg Ala Pro Arg Ser Pro Lys
Met Val Gln Gly Ser Gly Cys 100 105 110Phe Gly Arg Lys Met Asp Arg
Ile Ser Ser Ser Ser Gly Leu Gly Cys 115 120 125Lys Val Leu Arg Arg
His 1303126PRTHomo sapiens 3Asn Pro Met Tyr Asn Ala Val Ser Asn Ala
Asp Leu Met Asp Phe Lys1 5 10 15Asn Leu Leu Asp His Leu Glu Glu Lys
Met Pro Leu Glu Asp Glu Val 20 25 30Val Pro Pro Gln Val Leu Ser Asp
Pro Asn Glu Glu Ala Gly Ala Ala 35 40 45Leu Ser Pro Leu Pro Glu Val
Pro Pro Trp Thr Gly Glu Val Ser Pro 50 55 60Ala Gln Arg Asp Gly Gly
Ala Leu Gly Arg Gly Pro Trp Asp Ser Ser65 70 75 80Asp Arg Ser Ala
Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala 85 90 95Pro Arg Ser
Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg 100 105 110Ile
Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 115 120
1254151PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Met Ser Ser Phe Ser Thr Thr Thr Val Ser Phe
Leu Leu Leu Leu Ala1 5 10 15Phe Gln Leu Leu Gly Gln Thr Arg Ala Asn
Pro Met Tyr Asn Ala Val 20 25 30Ser Asn Ala Asp Leu Met Asp Phe Lys
Asn Leu Leu Asp His Leu Glu 35 40 45Glu Lys Met Pro Leu Glu Asp Glu
Val Val Pro Pro Gln Val Leu Ser 50 55 60Asp Pro Asn Glu Glu Ala Gly
Ala Ala Leu Ser Pro Leu Pro Glu Val65 70 75 80Pro Pro Trp Thr Gly
Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala 85 90 95Leu Gly Arg Gly
Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys 100 105 110Ser Lys
Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser 115 120
125Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu
130 135 140Gly Cys Asn Ser Phe Arg Tyr145 1505126PRTHomo sapiens
5Met His Leu Ser Gln Leu Leu Ala Cys Ala Leu Leu Leu Thr Leu Leu1 5
10 15Ser Leu Arg Pro Ser Glu Ala Lys Pro Gly Ala Pro Pro Lys Val
Pro 20 25 30Arg Thr Pro Pro Ala Glu Glu Leu Ala Glu Pro Gln Ala Ala
Gly Gly 35 40 45Gly Gln Lys Lys Gly Asp Lys Ala Pro Gly Gly Gly Gly
Ala Asn Leu 50 55 60Lys Gly Asp Arg Ser Arg Leu Leu Arg Asp Leu Arg
Val Asp Thr Lys65 70 75 80Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln
Glu His Pro Asn Ala Arg 85 90 95Lys Tyr Lys Gly Ala Asn Lys Lys Gly
Leu Ser Lys Gly Cys Phe Gly 100 105 110Leu Lys Leu Asp Arg Ile Gly
Ser Met Ser Gly Leu Gly Cys 115 120 125
* * * * *
References